text:
EGCG Enhances Cisplatin Sensitivity by Regulating Expression of the Copper and Cisplatin Influx Transporter CTR1 in Ovary Cancer

Cisplatin is one of the first-line platinum-based chemotherapeutic agents for treatment of many types of cancer, including ovary cancer. CTR1 (copper transporter 1), a transmembrane solute carrier transporter, has previously been shown to increase the cellular uptake and sensitivity of cisplatin. It is hypothesized that increased CTR1 expression would enhance the sensitivity of cancer cells to cisplatin (cDDP). The present study demonstrates for the first time that (-)-epigallocatechin-3-gallate (EGCG), a major polyphenol from green tea, can enhance CTR1 mRNA and protein expression in ovarian cancer cells and xenograft mice. EGCG inhibits the rapid degradation of CTR1 induced by cDDP. The combination of EGCG and cDDP increases the accumulation of cDDP and DNA-Pt adducts, and subsequently enhances the sensitivity of ovarian cancer SKOV3 and OVCAR3 cells to the chemotherapeutic agent. In the OVCAR3 ovarian cancer xenograft nude mice model, the combination of the lower concentration of cDDP and EGCG strongly repressed the tumor growth and exhibited protective effect on the nephrotoxicity induced by cisplatin. Overall, these findings uncover a novel chemotherapy mechanism of EGCG as an adjuvant for the treatment of ovarian cancer.

Introduction
Ovarian cancer is the seventh most common cancer (estimated age-standardized incidence and mortality) in women worldwide and is one of the leading causes of mortality among gynecological malignancies [1]. In recent decades, even though new therapeutic strategies have been developed, surgery and platinum-based chemotherapy are still the standard treatments for ovarian cancer [2].
Cisplatin (cis-diamminedichloroplatinum, cDDP) is one of the first-line chemotherapeutic agents in the treatment of ovarian cancer. cDDP exerts its cytotoxic effect predominantly by formatting an intra-strand cross-linking on DNA that blocks transcription and DNA replication, resulting in cell apoptosis [3]. However, drug resistance is an important limitation in the clinical application of cDDP. Mechanisms of cDDP resistance are complicated, including decreased drug uptake, increased drug efflux, increased DNA damage repair and alterations in apoptotic signaling pathways [4]. Recent studies suggested that copper transporters not only are involved in copper homeostasis but also regulate the cellular pharmacology and sensitivity to platinum-based agents [5,6].
The family of copper transporters consists of copper transporters and copper transporting phosphorylated ATPase (ATP7A and ATP7B). The former includes transmembrane solute carrier transporter CTR1 (encoded by SLC31A1) and CTR2 which regulate the influx of platinum-containing agents. CTR1 contains 190 amino acids and has three transmembrane domains. The N-terminal extracellular domain is rich in methionine and histidine, and is essential for transporting copper [7,8]. Several studies indicated that CTR1 is a major regulator of the efficacy of platinum drugs in vitro and in vivo [9,10]. Over-expression of CTR1 was found to sensitize cells to platinum drugs by increasing drug uptake [11,12]; whereas knockdown of CTR1 rendered cells resistant to these agents in yeast and several mammalian cells [13][14][15]. CTR1 could be rapidly degraded by cDDP, even at a low concentration (2μM) [15]. This process involves ubiquitination and proteasomal degradation and requires the copper chaperone antioxidant protein 1 (ATOX1) [16]. Unlike CTR1, CTR2 decreases cellular cDDP accumulation and sensitivity to cDDP of several types of tumors [17,18]. Knock-down of CTR2 leads to increased concentration of cDDP and this effect is opposite to knock-down of CTR1 [19]. ATP7A and ATP7B facilitate the sequestration and export of platinum-containing agents and are two promising chemoresistance markers for cDDP in various solid tumors [20,21].
(-)-Epigallocatechin-3-gallate (EGCG) is the most abundant and active polyphenol found in green tea and has been extensively studied in cancer prevention and therapy [22,23]. It has been found that EGCG has anti-cancer effect in several cancer types including ovarian cancer. The anti-cancer effect of EGCG may involve its inhibition of cancer process during the initiation, progression and metastasis [24].
There is a large body of evidence suggests that EGCG enhances the effect of conventional cancer therapies in multiple cell models [25]. In the case of the combination of EGCG and cDDP, EGCG can enhance the sensitivity of cDDP via multiple mechanisms, including up-regulating caspase-9a [26], potentiating G2/M arrest and up-regulating p21 [27], as well as inducting of apoptosis through enhanced intracellular H 2 O 2 generation [28]. EGCG treatment also inhibits telomerase expression and makes cells more sensitive to cDDP [29]. A more recent study found that EGCG suppressed ABCC2 and ABCG2 transporter genes, then augmented the efficacy of cDDP [30]. As a chemosensitizer, EGCG regulates microvasculature and microenvironment and improved cDDP sensitivity by rebalancing Ang-1 and Ang-2 [31]. In lung cancer cells, EGCG could enhance the efficacy of cDDP by down-regulating hsa-miR-98-5p [32].
Although the mechanisms of action of EGCG in combination with cDDP have been intensively studied, there is no research focused on the uptake of cDDP. Here, we investigated whether EGCG could modulate the copper transporters involved in the uptake and efflux of cDDP in vitro and in vivo. We analyzed the effect of EGCG in combination with cDDP on cells sensitivity to cDDP, the accumulation of cDDP and DNA-cDDP adducts, and the expression of copper transporters in ovarian cancer cells. Interestingly, we made a novel observation that EGCG induced the expression of CTR1 in vivo and in vitro. The findings provides experimental evidence for considering the application of EGCG as an adjuvant in ovarian cancer therapy.

Cell culture and reagents
Human ovarian cancer OVCAR3, SKOV3 cells and human embryonic kidney HEK-293T cells were obtained from Chinese Academy of Sciences Committee on Type Culture Collection Cell Bank (Shanghai, China). All these cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, GIBCO, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (GIBCO, Carlsbad, CA, USA), and 100U/ml penicillin and 100mg/ml streptomycin. Cells were incubated at 37°C in a humidified atmosphere of 5% CO 2 . EGCG and cDDP were purchased from Sigma-Aldrich (St. Louis, MO, USA).

MTT assay
MTT assay was used to evaluate cell viability. Cells were seeded in a 96-well plate at a density of 2 × 10 3 per well and allowed to attach overnight. Then EGCG alone or in combination with cDDP was administrated in a final volume of 200 μl of medium. After treatment for 24 h or 48 h, the cells were incubated at 37°C with 20 μl of MTT solution (5 mg/ml,Amresco, OH, USA) for 4 h. The MTT formazan crystal was then dissolved in 150 μl DMSO (Lingfeng, Shanghai, China), and a microplate reader (Tecan, Mannedorf, Switzerland) was used to measure the absorbance at 490 nm.

Colony formation assay
After indicated treatment, the cells (2 × 10 2 ) were seeded in a 6-well plate and changed to a fresh medium every 3 days. After three weeks, visible colonies were fixed and stained with crystal violet staining solution (Beyotime, Shanghai, China).

Hoechst 33258 staining
The cells were seeded in a 6-well plate. After incubation for 24 h, EGCG or cDDP was added alone or in combination for 48 h. Then the cells were fixed in 4% paraformaldehyde for 10 min, and washed twice with PBS. After being stained with 0.5 ml of Hoechst 33258 (Beyotime, Shanghai, China) for 5 min, the cells were again washed twice with PBS. The stained nuclei were observed under an inverted fluorescence microscopy (Olympus, Japan).

Measurement of platinum (Pt) accumulation in cells
Whole-cell Pt content was measured by a Perkin-Elmer Element 2 inductively coupled plasma mass spectrometry (ICP-MS) according to previously reported methods [17]. For measurement of Pt in DNA, the DNA was extracted using DNAzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. For normalization, the concentration of DNA was measured using a Nanodrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). The DNA samples were then digested in 5% nitric acid and measured by ICP-MS.

Reverse Transcription Polymerase Chain Reaction (RT-PCR)
Total RNA was isolated from cells using the RNAiso Plus (TaKaRaBioTechnology, Dalian, China) following the manufacturer's protocol. The concentration of total RNA was measured using a Nanodrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE). Total RNA was reverse transcribed using the PrimeScriptRT Master Mix (TaKaRaBioTechnology), and PCR was performed using Premix Taq (TaKaRaTaq Version 2.0 plus dye, TaKaRaBioTechnology). The mRNA specific primers of CTR1, CTR2, ATP7A and ATP7B were purchased from Genscript Corp. (Nanjing, China). The PCR products were separated by electrophoresis in 2% agarose gels and were detected using the AlphaImager HP system (Alpha Innotech, San Leandro, CA, USA).

Western blotting
After the indicated treatment, the cells were harvested and lysed by RIPA buffer (KeyGENBio-TECH, Nanjing, China). The protein was extracted and its concentration was measured by BCA Protein Assay Kit (Beyotime, Shanghai, China). SDS-PAGE was used to separate 60 μg of protein. Then followed by transfer onto a PVDF membrane (Millipore Corporation, MA, USA). Membranes were blocked for 1 h at room temperature with 5% non-fat dry milk in Tris-Buffered-Saline with Tween (TBST), followed by incubation overnight at 4°C with specific antibodies. After being washed 3 times for 5 min with TBST, the membrane was then incubated with appropriate secondary antibodies for 1 h at room temperature. After extensive washing with TBST, proteins were visualized by the SuperSignal West Pico (Thermo Scientific, Wilmington, DE, USA). Antibodies used include: anti-CTR1 (1:1000, Santa Cruz Biotechnology, CA, USA), anti-β-actin (1:1000

Ethics statement
This study was carried out strictly with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of Nanjing medical university (Permit Number: 2120474).

Xenograft mouse model
Female BALB/c nude mice, 3-5 weeks of age, were purchased from Shanghai Animal Laboratory Center (Shanghai, China) and maintained in appropriate sterile filter-capped cages with lights turned on at 8:30 am in the Experimental Animal Center at Nanjing Medical University. The temperature and humidity was 22 ± 1°C and 55 ± 5%. We put wood shavings, bedding and a cardboard tube in all cages for environmental enrichment. All mice were observed every day to monitor abnormal behavior, such as inability to eat or drink, no response when stimulated, or unable to run away when touched. Exponentially growing OVCAR3 cells (5 × 10 6 ) were injected subcutaneously into the dorsum of the mice. After tumor transplantation for 1 week, the body weight and the tumor size were recorded twice a week. The length and width of tumor were measured using a caliper, and the volumes were calculated by the following formula: volume (mm 3 ) = length × width × width/2. At the 18th day after transplantation, 32 mice were randomized into 4 groups (6 mice of control group and 8 mice of the other group) and treated as follows: control (normal saline, 0.1ml/10g), EGCG (20 mg/kg), cDDP (5 mg/kg), EGCG (20 mg/kg) and cDDP (5 mg/kg). EGCG was administered twice a week and cDDP was given once a week by intraperitoneal injection. After treatment for 4 weeks, xenograft tumors and kidney tissue were isolated from mice. A portion of the tumors and kidney tissue were fixed in 4% paraformaldehyde for histological study, and the rest were prepared for further experiments. We tried our best to minimize suffering of the Female BALB/c nude mice. No mice were sacrificed before the end of the experiment and the mice were euthanized by cervical dislocation.

Renal damage indicators
Renal damage was measured with blood urea nitrogen (BUN) and plasma creatinine. BUN was measured by diagnostic kits (Jiancheng Bioengineering Institute, Nanjing, China) according to the assay kit protocols. We used urease methods to measure the BUN and picric acid methods to test the serum creatintine.

Statistical methods
All data were presented as the mean ± standard deviation (SD) of at least three independent experiments. Western blotting was quantified using Image J software. Statistical analysis was performed using the student's t test and One-Way ANOVA. The data were considered statistically significant when the P value was less than 0.05. ( Ã P<0.05, ÃÃ P<0.01)

EGCG enhances the sensitivity of the ovarian cancer cells to cDDP
To determine the suitable concentration of EGCG on cells survival, ovary cancer OVCAR3 and SKOV3 cells were treated with indicated doses of EGCG. As Fig 1A showed, from 10 μM, EGCG caused significantly inhibition of the cells growth. Therefore, 10 μM EGCG was chosen for combination with cDDP. To determine the effect of EGCG on cDDP sensitivity, OVCAR3 and SKOV3 cells were treated with 10μM EGCG and various concentration of cDDP, either alone or in combination. As shown in Fig 1B, cDDP alone inhibited the growth of the ovary cancer cells in a dose-dependent manner, while the combination of EGCG and cDDP exhibited a greater inhibition. In OVCAR3 cells, the IC 50 (mean ± SEM) was 9.49 ± 0.13 μM for cDDP alone and 3.48 ± 0.24 μMcDDP for the combination. The combination decreased IC 50 for 2.7-fold in SKOV3 cells. The IC 50 had a 1.6-fold decrease in the combination of EGCG and cDDP (6.14 ± 0.38 μM), compared with cDDP (9.65 ± 0.56 μM) only treatment.
To further confirm the effect of EGCG and cDDP on cell growth, a colony formation assay was conducted. EGCG or cDDP inhibited colony growth, while the combination of the two conferred a more pronounced repression (Fig 1C). The possible induction of apoptosis in OVCAR3 cells by EGCG and cDDP was investigated by Hoechst 33258 staining. The combination of the two agents showed significant apoptosis in OVCAR3 cells (Fig 1D).

EGCG increases cDDP and DNA-Pt adducts accumulation in cells
To investigate whether changes in sensitivity to cDDP were resulted from changes in cellular cDDP and DNA-Pt accumulation, the whole-cell content of Pt was measured by ICP-MS. Fig  2A showed that the OVCAR3 cells exposed to 30 μM of cDDP for 4 h accumulated 29.57±2.52 ng of Pt per 1 μg protein; whereas the cells treated with combined EGCG and cDDP accumulated 40.04±2.78 ng of Pt per 1 μg protein, representing a 35% elevation as compared with the cDDP group (P<0.01).
The primary mechanism of action of Pt-containing drugs is thought to format DNA-Pt adducts and cause cytotoxicity. Thus, the DNA-Pt adduct concentration was measured in DNA extracted from cells with 30μMof cDDP or cDDP combined with 10 μM of EGCG. The combination of the two agents caused a 42% increase in the DNA-Pt accumulation (7.66±0.70 versus 5.41±0.46 μg Pt/μg DNA; P<0.01) (Fig 2B). Therefore, the observation of EGCG enhancing cDDP sensitivity might be partly explained by a proportional increase in the amount of the cDDP and DNA-Pt accumulation in the cells.

EGCG promotes the expression of CTR1 but not the other copper transporters
Given the fact that the copper transporters can partly influence the sensitivity of ovarian cancer cells to cDDP, the relative expression of copper transporters was quantified by RT-PCR and qPCR. As shown in Fig 3A, CTR1 mRNA was elevated after EGCG treatment in OVCAR3 cells. However, there were no significant changes in mRNA expression of CTR2, ATP7A and ATP7B upon EGCG treatment. Next, we focused on whether EGCG could affect CTR1 protein expression. As shown in Fig 3B, EGCG was able to induce CTR1 protein expression in a dosedependent manner either in OVCAR3 and SKOV3 cells (Fig 3C). Taken together, EGCG can induce CTR1 expression in ovary cancer cells.

Knock-down of CTR1 changes the sensitivity of cells to cDDP
To further confirm the relationship between CTR1 and cDDP sensitivity, OVCAR3 cells were transfected with CTR1 siRNAs. The si-RNA3 shows the best knockdown effect. The expression of CTR1 (Fig 4A and 4B) and the survival fraction of cells responding to cDDP were measured. As shown in Fig 4A and 4B, knockdown of CTR1 decreased the expression of CTR1 and lowered the sensitivity of ovarian cancer OVCAR3 and SKOV3 cells to cDDP. Interestingly, knockdown of CTR1 had no statistically significant effect in human embryonic kidney HEK-293T cells, though the expression of CTR1 was almost completely inhibited. These results indicated that the influence of CTR1 to cDDP might be different between cell types.

EGCG inhibits the degradation of CTR1 induced by cDDP
Previous studies have shown that, even at lower concentrations, cDDP can induce rapid degradation of CTR1 [15]. As shown in Fig 5A and 5B, 10 μM of cDDP decreased the expression of CTR1 in a time-dependent manner in OVCAR3 and SKOV3 cells. To determine the contribution of proteasomal degradation to the down-regulation of CTR1 induced by cDDP, OVCAR3 cells were incubated with cDDP alone or in combination with MG132 (a proteasome inhibitor), western blot analysis indicated that MG132 blocked the degradation of CTR1 (Fig 5C). Since EGCG could induce the expression of CTR1, we next determined whether EGCG could prevent the degradation of CTR1 induced by cDDP. Fig 5D and 5E showed that EGCG inhibited the rapid degradation of CTR1 caused by cDDP treatment in ovarian cancer OVCAR3 and SKOV3 cells.

EGCG enhances the efficacy of cDDP in inhibiting xenograft tumor growth and attenuates the nephrotoxicity of cDDP in vivo
To further investigate whether the enhanced efficacy of cDDP by EGCG could occur in vivo, we established an OVCAR3 ovarian xenograft model in nude mice as described in the Materials and Methods section. The body weight and tumor size were measured twice weekly. The body weights of the cDDP-treated groups were significantly lower compared with the other groups ( Fig 6A). The administration of EGCG exhibited a protective effect against the cDDP-or in combination with the EGCG (10 μM, shown as E10), and followed by MTT assay. (C) EGCG in combination with cDDP repressed colony formation. OVCAR3 cells were treated with 10 μM of EGCG alone or in combination of 10 μM of cDDP for 48 h. When the colonies formed two weeks later, colony formation assay was carried out. (D) The combination of EGCG and cDDP on cells apoptosis, Hoechst 33258 staining was used to detect apoptosis caused by the indicated treatments. (*P<0.05, **P<0.01) doi:10.1371/journal.pone.0125402.g001  Fig 6A, EGCG or cDDP alone suppressed the growth of tumor, while EGCG combined with cDDP had a stronger inhibitory effect than other treatments, which was consistent with the results in vitro experiments.
The mRNA and protein expression of CTR1 were measured in tumor tissues ( Fig 6B). As it was shown, EGCG increased the mRNA expression of CTR1 significantly. It also appeared that the protein expression of the CTR1 was significantly decreased by cDDP, but enhanced by EGCG and returned to a normal level by EGCG combined with cDDP ( Fig 6C). The renal damage indicators such as kidney weight, blood urea nitrogen (BUN) and plasma creatinine were used to evaluate possible renal damage. The mice treated with cDDP, their kidney weights, BUN and creatinine were significantly higher than those of other groups. Administration of EGCG reduced the levels of these cDDP-induced kidney toxicity indicators. Treatment with EGCG alone did not affect these indicators (Table 1). All these results demonstrated that EGCG could partly protect against nephrotoxicity induced by cDDP in vivo. To investigate whether the protective effect of EGCG on cDDP-induced nephrotoxicity was associated with the uptake of cDDP, we measured the relative expression of CTR1 in kidney tissues. The expression of CTR1 had no significant changes in kidney tissues after the indicated treatments ( Fig 6D).

Discussion
In the current study, we systematically investigated the effects of EGCG on copper transporters and the uptake and efflux of cDDP in ovary cancer cells and xenograft mice. To our knowledge, this is the first report demonstrating that EGCG increased CTR1 expression in vitro and in vivo. Also, EGCG prevented the degradation of CTR1 induced by cDDP. EGCG treatment in ovarian cancer resulted in a significant increase in sensitivity to cDDP, which might be due to   the up-regulation of CTR1, increased accumulation of cDDP and DNA-Pt adducts. Our findings provided a mechanism for the strategy of using EGCG as an adjuvant for ovarian cancer therapy.
Previous studies showed that the expression of CTR1 could be regulated under different copper stress at the transcriptional and post-translational levels. At the transcriptional level, the mRNA of CTR1 was regulated by the transcription factor Sp1 [33]. The zinc finger (ZF) domain of Sp1 was found to function as sensors of copper stress [33], and there existed a Copper-hCtr1-Sp1 Inter-Regulatory Loop in copper homeostasis [34]. Post-translational mechanisms have also been related to copper stress. Copper chelator or copper-lowering agents were shown to induce CTR1 expression and enhance the uptake of cDDP by cells and their sensitivity to cDDP [10,35,36]. High concentrations of copper and cDDP induced endocytosis or degradation of CTR1 [15,37,38]. The mechanism through which EGCG mediates CTR1 expression is still not understood. More studies on this topic are needed.
In the present study, cDDP(in 10 μM) caused rapid degradation of CTR1 (Fig 5A and 5B). Multiple lines of evidence indicated that cDDP-induced degradation of CTR1 involves ubiquitination and proteosomal degradation. A proteasome inhibitor (bortezomib, actacystin or MG132) given together with cDDP blocked cDDP-induced loss of CTR1 and enhanced delivery of cDDP [39][40][41]. Combination of cDDP with EGCG or MG132 both inhibited the degradation of CTR1 induced by cDDP (Fig 5C, 5D and 5E). It was reported that EGCG could inhibit the proteasome activity [22,42]. The inhibition of ubiquitin-proteasome by EGCG contributed to overcome resistance to various chemotherapeutic drugs and enhance the effect of chemotherapy [43]. Thus, the inhibition of cDDP-induced degradation of CTR1 by EGCG might be due to its action on ubiquitin-proteasome.
Nephrotoxicity is the major complication of the clinical application of cDDP and limits its long-term usage. Previous studies have reported that EGCG could prevent the deleterious effects of cDDP [44][45][46]. Our study also indicated that EGCG had a protective effect against cDDP-induced nephrotoxicity ( Table 1). One of the reasons could be that EGCG didn't change CTR1 expression in kidney tissues (Fig 6), while EGCG induced CTR1 expression in ovarian cancer cells and tumor tissues (Figs 3 and 6). Knockdown of CTR1 in human embryonic kidney HEK-293T cells had on significant changes to cDDP sensitivity ( Fig 4B). Why EGCG did not change CTR1 expression in kidney cells and tissues is interesting and needs further investigation.
The protective effect of EGCG against nephrotoxicity may be also due to its antioxidant and anti-inflammation activities as suggested previously [44][45][46]. Recent studies suggested that  organic cation transporter 2 (OCT2) was a target for protective interventions in cDDP-induced nephrotoxicity [47][48][49]. Our preliminary data showed that EGCG could regulate OCT2 as well, and this is under further investigation. In summary, this is the first report that EGCG could induce the expression of CTR1, leading to the accumulation of cellular cDDP and cDDP-DNA adducts and enhanced sensitivity of ovarian cancer cells to cDDP. The findings provided a novel mechanism for exploring new strategies for green tea polyphenol EGCG as an adjuvant for the treatment of ovarian cancer.

Author Contributions
Conceived and designed the experiments: Xuemin Wang QF. Performed the experiments: Xuemin Wang PJ PW. Analyzed the data: C. Yang Xuerong Wang. Contributed reagents/materials/analysis tools: C. Yang Xuerong Wang QF. Wrote the paper: Xuemin Wang QF.


s2fieldsofstudy: Biology Medicine

text:
Link between chronic inflammation and human papillomavirus-induced carcinogenesis (Review)

Inflammation is a defense strategy against invading agents and harmful molecules that is activated immediately following a stimulus, and involves the release of cytokines and chemokines, which activate the innate immune response. These mediators act together to increase blood flow and vascular permeability, facilitating recruitment of effector cells to the site of injury. Following resolution of the injury and removal of the stimulus, inflammation is disabled, but if the stimulus persists, inflammation becomes chronic and is strongly associated with cancer. This is likely to be due to the fact that the inflammation leads to a wound that does not heal, requiring a constant renewal of cells, which increases the risk of neoplastic transformation. Debris from phagocytosis, including the reactive species of oxygen and nitrogen that cause damage to DNA already damaged by the leukotrienes and prostaglandins, has an impact on inflammation and various carcinogenic routes. There is an association between chronic inflammation, persistent infection and cancer, where oncogenic action is mediated by autocrine and paracrine signals, causing changes in somatic cells under the influence of the microbial genome or of epigenetic factors. Among the infectious agents associated with cancer, certain genotypes of human papillomavirus (HPV) stand out. HPV is responsible for virtually all cases of cervical cancer and a lower proportion of cancers of the vagina, vulva, anus, penis and a number of extragenital cancers. In the present review, recent advances in the mechanisms involved in the inflammatory response are presented with their participation in the process of carcinogenesis, emphasizing the role of chronic inflammation in the development of HPV-induced cervical cancer.

Introduction
Inflammation is a universal phenomenon that works during the occurrence of disturbances in homeostasis caused by infections, injury and exposure to irritants. The inflammatory reaction begins when molecular signaling pathways associated with pathogens or tissue damage are detected by the receptors of the innate immune system (1). A distinctive characteristic of the inflammatory response, in relation to other mechanisms of defense, is that during inflammation, damage to the host is inevitable. Despite the collateral damage that can result in pathologies, inflammation seeks the balance between tissue damage and self-maintenance (1,2). The challenge is to balance the immune response, so that the offending agent is removed with minimal damage, which is known as disease ecology (3).
From the physiological point of view, the function of inflammation is to quickly destroy or isolate the source of

Link between chronic inflammation and human papillomavirus-induced carcinogenesis (Review)
the disturbance, repair the damage incurred and restore homeostasis, being, therefore, a natural adaptation. One piece of evidence for this is the increased risk of serious infections in humans with genetic disorders in the primary components of inflammation. The same occurs in animals with deficient expression of genes encoding pro-inflammatory cytokines (4). By contrast, the deregulation of immunologically relevant genes leads to spontaneous inflammation, suggesting that, under normal conditions, the inflammatory response is suppressed to maintain health (5). The lack of control leads to exaggerated inflammation that can have devastating effects and cause severe pathologies (1).
Acute inflammation is quickly resolved following the removal of the stimulus and the repair of the tissue, but if the disturbance persists for a long time without repair, inflammation becomes chronic. This requires a constant cell renewal, which, in turn, leads to the acceleration of cell division and an increased risk of mutation and neoplastic transformation. Persistent infection induces chronic inflammation, where products generated by leukocytes and other cells, including leukotrienes and reactive species of oxygen and nitrogen, can cause DNA damage. Inflammatory cells also release prostaglandins produced by the action of enzyme cyclooxygenase-2 (COX2), which intensifies the inflammation and has an impact on various carcinogenic routes (6).
Clinical and epidemiological studies have revealed that certain pathogens that cause persistent infections are strongly associated with cancer (6,7). The oncogenic action appears to be mediated by autocrine and paracrine signals that are mediated by chronic inflammation, or by somatic alterations in the cells induced by the microbial genome (8).
The persistent infection with high-risk human papillomavirus (HR-HPV) is clearly defined as necessary for invasive cervical cancer (ICC) development. Once integrated with the host cell genome, HPV can cause genetic rearrangements, deletions, chromosomal inversions and translocations, activation of proto-oncogenes and loss of heterozygosity, which generates genomic instability and increases the risk of neoplastic transformation (9). However, the infection itself is not sufficient for transformation of the infected cell, unless it is accompanied by additional epigenetic events, including inflammation, facilitated by exposure to environmental factors or due to altered immunological mechanisms (10)(11)(12)(13)(14). In the present review, the recent advances about of the molecular and immunological mechanisms involved in the inflammatory reaction are reported, as well as the role of chronic inflammation in the development of HPV-associated cancer.
The literature search was conducted using the electronic databases PubMed (National Institutes of Health, Bethesda, MD, USA; www.ncbi.nlm.nih.gov/pubmed), Scopus (Elsevier, Amsterdam, Netherlands; www.scopus.com/scopus/home.url) and Web of Knowledge (Thomson Reuters, New York, NY, USA; wok.mimas.ac.uk), using the following keywords: Carcinogenesis, inflammation, inflammation and cancer, inflammation and HPV infection, and inflammation and cervical cancer. The databases retrieved thousands of articles and those that were thought to be most relevant were selected, which included recent studies published in journals of impact, conducted by groups with recognized expertise in the areas of inflammation, cancer and infectious agents, in particular HPV.

Mediators of the inflammatory response
Inflammation is a non-specific defense mechanism activated in response to tissue damage of any nature, and involves the innate and adaptive immune system combating the aggressor agents (15). This process consists of a complex cascade of events involving the participation of soluble mediators, termed cytokines and chemokines, which act collectively in the activation and regulation of the inflammatory response. Table I presents the main cytokines involved in the inflammatory response, the cells that produce them and the main mechanisms through which each operates during the activation and regulation of the inflammatory reaction .

Molecular mechanisms of the inflammatory response
Inflammation is a biological reaction that aims to correct disturbances in homeostasis occurring in the tissues by recruiting of leukocytes and other blood products, such as plasma proteins, to the site of injury. This migration is facilitated by vasodilation and increased vascular permeability and blood flow. Once activated, proinflammatory cytokines, including tumor necrosis factor (TNF), interleukin (IL)-1β and IL-6 are produced, but once the stimulus is removed, inflammation becomes naturally regulated and returns to its normal state (39). The reversion of inflammation to the state of homeostasis precedes a highly-regulated process, known as the resolution of inflammation, which is controlled by various anti-inflammatory mediators, particularly IL-10, transforming growth factor (TGF)-β and glucocorticoids, involving the recruitment of monocytes for the cleaning of cell debris (40).
The inflammatory response is a tightly regulated cascade of immunological, physiological and behavioral processes. The activation of inflammation begins when pattern recognition receptors (PRRs), which are expressed in the cells of the innate immune system, detect the presence the conserved structures of harmful agents, termed pathogen-associated molecular patterns (PAMPs), or endogenous molecules derived from lesions, termed damage-associated molecular patterns (DAMPs) (41,42). The PRRs are transmembrane receptors of various types, including Toll-like receptors (TLRs), C-type lectin receptors, RIG-like receptors, nucleotide-binding oligomerization domain-like receptors and rich-leucine receptors, which have a selective ability to detect PAMPs, DAMPs or the two together (43,44).
Once they have recognized PAMPs or DAMPs, the PRRs bind to target molecules and trigger common signaling pathways, culminating in the activation of nuclear factor κ of B cells (NF-κB), which is transported to the nucleus of the cell. In the nucleus, NF-κB activates the transcription of various genes, whose products instantly trigger the inflammatory response through the production of cytokines, chemokines and other mediators involved in the inflammatory process (42,45). In the case of viral infections, the signaling pathway involves the use of interferon type 1 (IFN-1) and the activation of cytotoxic T lymphocytes (CTLs). IFN-1 induces the phosphorylation and transport to the nucleus of a complex termed interferon-stimulated gene factor 3, which is composed of signal transducers and activators of transcription (STAT)-1 and -2, and IFN regulatory factor 3 (IRF-3). In turn, this activates the expression of antiviral genes, including protein kinase R (PKR) and 2'-5'-oligoadenylate synthetase (OAS). Thus, the proliferation of virus-infected cells is inhibited by PKR, while OAS suppresses viral replication by the cleavage of nucleotides (46,47).
Soluble factors, including IL-6 and macrophage colony-stimulating factor-1 (CSF-1), derived from neoplastic cells, induce differentiation in myeloid cells to produce a similar phenotype as a macrophage. These cells are known as tumor-associated macrophages (TAMs) and they are the major components of inflammatory infiltrates in tumor tissue, and are recruited by monocyte chemotactic protein and other mediators (48). M2-polarized TAMs are key regulators of the link between inflammation and cancer. However, these cells have a contradictory role in neoplasia. TAMs can kill tumor cells following activation by IL-2, IFN-γ and IL-12, but they also produce IL-10, which inhibits the CTL-mediated antitumor response and therefore promotes tumor progression (5,48,49). Table I. Main cytokine mediators, the inflammatory response and their respective roles.

Cytokine Producing cell
Main functions

IL-1β
Monocytes and IL-1β exhibits proinflammatory action, participates in the recruitment and activation of the macrophages cells of the adaptive immune system and acts as a signal for the activation of nuclear factor kappa of B cells (NF-κB) (16)(17)(18). TNF-α Inflammatory and TNF-α stimulates the growth and survival of tumor cells, exhibits antiapoptotic action, facilitates tumor cells tumor progression, promotes angiogenesis and decreases immune surveillance by suppressing the cytotoxic activity of T lymphocytes and activated macrophages (19). TGF-β Macrophages, TGF-β exhibits anti-inflammatory and immunosuppressive action, presenting a central role in T lymphocytes the proliferation and regulatory function of Treg cells by inducing Fox3. TGF-β also inhibits T-cell proliferation, the activation of macrophages and blocks the effects of proinflammatory cytokines (20,21). IL-6 Macrophages, IL-6 exhibits proinflammatory action and plays a central role in the adaptive immune response, T lymphocytes in inflammation and in tumor progression due to its ability to promote cell proliferation, inhibit apoptosis and neutralize the cytostatic effects of TGF-β (22,23).

IL-10
Macrophages IL-10 inhibits the production of IFN-γ by Th1 cells and the production of cytokines by activated and Th2 cells macrophages, inhibits the effects of IL-6 and IL-12. IL-10 also possesses antinflammatory and antiproliferative activity, therefore inhibiting the formation and progression of tumors (24,25).

IFN-α/β
Many cell types IFN-α/β are key effectors of the innate immune response to viral infections, activating a signaling cascade that activates numerous genes whose products inhibit viral replication. IFN-α/β also regulate the production of IFN-γ and possess anti-inflammatory activity, through inhibiting the production of IL-1, -18 and -12 and increasing the production of IL-10 (26-28). IFN-γ APCs, NK, NKT IFN-γ Activates macrophages during the innate immune response, increases the activity of and Th1 cells NK cells and the production of MHC class II by macrophages. Following the initiation of the adaptive immune response, IFN-γ upregulates the Th1-cell response and inhibits the response of Th2 cells (29,30).

IL-17
Th17 IL-17 plays a central role in the elimination of pathogens, but persistent production causes chronic inflammation, including the production of TNF-α, IL-1β and IL-6, which amplifies the inflammation. IL-17 is found in high levels in patients with cervical cancer (31)(32)(33).

IL-12
Activated APCs IL-12 is involved in the differentiation of naive T cells into Th1, stimulates the production of IFN-γ and TNF-α by T and NK cells, reduces IL-4-mediated suppression of IFN-γ and increases the cytotoxic activity of NK and TCD8 + cells (34,35).

IL-23
Activated APCs IL-23 acts in a paracrine manner by inducing macrophage production of TNF-α, DC production of IL-12 and IL-17 production, which promotes the end stage of inflammation. Despite the similarities between IL-12 and IL-23, they follow divergent immune pathways and exert distinct effects on the development of tumors (34,35).
Chronic inflammation is due to the action of various inflammatory mediators, including TNF-α, IL-6 and IL-17, which suppress antitumor immunity and favor tumor progression. By contrast, cytokines, including TNF-related apoptosis-inducing ligand (TRAIL), IL-10 and IL-12, exert antitumor action. TRAIL exerts antitumor effects by inducing tumor cell apoptosis, IL-10 exerts antitumor effects through its anti-inflammatory action and IL-12 exerts antitumor effects by activating CTLs and natural killer (NK) cells and through the expression of cytotoxic mediators, which can lead to tumor suppression (50). Thus, cytokines secreted by tumor and inflammatory/immune cells can either promote tumor development and tumor cell survival, or they can exert antitumor effects, depending on the context in which they operate. Upon infection by pathogens, activated myeloid cells produce proinflammatory and anti-inflammatory cytokines. In addition to its direct effects on tumor growth, survival of tumor cells and invasive properties, cytokines can govern the functions of T helper (Th)1 cells, NK cells, regulatory T cells (Tregs) and Th17 cells, all of which can infiltrate into tumors. Treg-mediated suppression of antitumor CTL responses and induction of inflammatory Th17 cell-mediated responses contribute to tumor progression. Paradoxically, IL-10 can mediate the antitumor effects of Tregs. IL-23, TGF-β, IL-6 and TNF-α promote the development of Th17 cells, which play a central role in the coordination of chronic inflammatory responses and, thus, can contribute to the growth of tumors. IL-23 induces the release of IFN-γ and IL-12 from activated T cells, TNF-α and IL-12 from antigen presenting cells (APCs), and IL-17 from Th17 cells (51). However, it has been revealed that IL-23 inhibits IL-12 activity, particularly the activation of IFN-γ (51).
In contrast to innate immunity, whose role is well defined, little is known about the involvement of adaptive immunity in the inflammatory response, although it has been documented in several studies (52)(53)(54). The Th cells represent a specific population of lymphocytes of the adaptive immune response and are largely responsible for activating and regulating this response. When stimulated by APCs, Th cells that were never exposed to the antigen, termed naive T or Th0 cells, can differentiate into effector cells with a Th1, Th2, Th17 or Treg phenotype. The Th1 phenotype exhibits proinflammatory action, Th2 exhibits anti-inflammatory action, Treg exhibits regulatory action and Th17 is involved with intense inflammatory responses and autoimmunity (55). Th1 cells activate cellular immunity and induce a proinflammatory response against intracellular pathogens through the release of IFN-γ, which has potent antiviral properties and promotes differentiation of Th0 in Th1 cells. Th1 cells also secrete IL-2, IL-12 and TNF-α, all of which are important in mediating the response of delayed-type hypersensitivity and activation of macrophages (56).
By contrast, Th2 cells are important for humoral immunity by activating the proliferation of B lymphocytes, regulating allergic responses and protecting against infection by macroparasites such as helminthes (1). Th2 cells produce anti-inflammatory cytokines, including IL-4, -5, -10 and -13, which further stimulates the differentiation of Th0 to Th2, promotes alternative activation of macrophages and induces the differentiation of B lymphocytes, which produce and secrete antibodies, in plasma cells (56). Th2 cells also induce immunoglobulin class switching for immunoglobulin (Ig) E and eosinophil maturation, and negatively regulate the production of Th1 cytokines. Indeed, Th1 and Th2 responses are antagonistic and represent a balance between proinflammatory and anti-inflammatory mechanisms, where one can be selected and influence the outcome of infection (57). The ideal condition would be a balance between the Th1/Th2 phenotypes to promote elimination of the pathogen with minimal inflammation and damage to host tissues. Animal studies have revealed a limited capacity of the host to mount Th1 and Th2 responses that are each effective (1,56,57).
The notion of a simplified bimodal immune response to infection that activates the Th1 or Th2 phenotype was modified by the discovery of the existence of a cross-regulation by a T cell type, termed regulatory T cells (Treg). Treg cells are important to suppress the activation, proliferation and effector functions of various immune cells, including T lymphocytes, NK cells, B lymphocytes and APCs (57). Although the role of Treg in regulating the homeostasis of the immune system has not yet been elucidated, there is evidence that these cells appear to dampen Th1 and Th2 immune responses by the secretion of IL-10 and TGF-β. These cytokines have immunosuppressive action and inhibit the proliferation of Th1 and Th2 cells, presumably to minimize tissue damage. Treg cells also appear to play a role in mediating chronic infection, avoiding the inflammation of immune privileged organs (58). This indicates that the inhibition of Treg function results in an immunopathological response with damage to tissues. However, an increase of these cells can increase the chance of survival of the pathogen and, in certain cases, leads to long-term chronic infection (59). Thus, establishing a good balance between effector and regulatory functions so that the host can efficiently deal with the pathogen is the big challenge. Th17 cells represent a population of effector Th cells, which are largely involved in the elimination of extracellular microparasites, a process that requires an intense inflammatory response and is not properly dealt with by Th1 or Th2 cells (60).

Persistent infection, chronic inflammation and carcinogenesis
In its broader definition, inflammation is a process that involves events of tissue remodeling caused by changes in the functions of the cells of the vascular and immune systems. Such changes are orchestrated by specific molecular pathways that involve a number of cytokines, chemokines, growth factors and lipid mediators (61). Compelling evidence indicates that the majority of cancers arise from sites of chronic irritation, infection and long-term inflammation, solidifying the concept that chronic inflammation decompensated is a critical factor for the progression of tumors (6).
The connection between chronic inflammation and cancer does not only operate in one direction. Studies have revealed that DNA damage may also result in inflammation, since inflammation and DNA damage induce cell death by necrosis, which leads to more inflammation (62,63). Oncoproteins that are associated with cancer, including Ras, Myc and RET, can also cause inflammation via the activation of signaling mechanisms involved in the production of proinflammatory cytokines and chemokines (64). The main molecular link between inflammation, promotion and tumor progression is provided by association with IK/NF-κB, involving a signaling pathway that is activated by numerous proinflammatory cytokines (65,66). NF-κB is a transcription factor that regulates the expression of several genes that may suppress cell death through apoptosis and the stimulation of cell cycle progression in tumor cells. In addition, NF-κB promotes epithelial mesenchymal transition, which plays an important role in invasiveness as it provides the emerging tumor with an inflammatory microenvironment that supports tumor progression and invasion of the surrounding tissue, causing angiogenesis and metastasis (67)(68)(69).
HPV is a sexually transmitted agent deemed a cause of cervical intraepithelial neoplasia (CIN) and invasive cervical cancer (ICC) worldwide. Currently it is widely accepted that specific genotypes of HPV are potentially oncogenic and are associated with virtually all cases of cervical cancer and, to a lesser extent, with cancers of the vagina, vulva, anus, penis, skin and oropharynx (10,(70)(71)(72). In general, HPV infection begins with viral replication in the cells of the germinal layer of the cervical epithelium. The viral replication continues in parallel with the differentiation of cells of the squamous epithelium, but assembly of virions only occurs in cells of the superficial layer of the epithelium that have reached the maximum degree of differentiation (73). In the majority of cases, the genital HPV infection is transient and heals spontaneously within 30 months. However, in certain individuals, the viral clearance does not occur during this period, the infection becomes persistent and can result in lesions that may eventually progress to cancer (74,75). Worldwide, cervical cancer is the second most common type of cancer in women, excluding non-melanoma skin cancer (76). The analysis of the pathogenesis of genital HPV infection and of the lesions associated with it, based on morphology, molecular biology, and epidemiology, revealed the existence of the infection without any lesion, with the presence of precursor lesions of low or of high-grade and invasive cancer (73). In the transition of HPV infection without lesions to cervical cancer, there is a gradual worsening of cytological changes characterized by increased nuclear atypia and a disorganized differentiation of the cervical epithelium, without invasion of the basement membrane, in the case of premalignant lesions or with invasion of the basement membrane in cases of cancer (75).
Persistent infection with HPV genotypes with high oncogenic potential increases the probability of ICC following an extended period of latency. The integrity of the cell-mediated immune response against HPV is an important factor for healing and prevention of the reactivation of the latent infection (75,77). Epidemiological studies in HPV-infected females have provided important clues about a spectrum of cofactors that can increase the carcinogenic potential of HPV. Presumably, cervical infections with other pathogens, exposure to physical and chemical agents, hormonal factors, nutritional status and genetic inheritance can serve as cofactors influencing the acquisition of HPV infection, as well as influencing the development of viral persistence (11,77). Certain studies suggest that other sexually-transmitted viral or bacterial agents may serve as cofactors for the development of HPV-associated cancer. These agents include the herpes simplex virus type 2 (HSV-2) (11,78) and Chlamydia trachomatis (79,80). Notably, the infections caused by these two pathogens are often associated with an intense chronic inflammatory response and ulcerations in the cervical epithelium (81,82).

microRNAs (miRNAs) inflammation and cancer
miRNAs are small non-coding RNAs that regulate the translation and degradation of target messenger RNAs (83). miRNAs have been implicated in the regulation of almost all aspects of cellular functions, including the innate and adaptive immune responses (84). miRNAs participate in numerous regulatory networks of genes whose dysfunctions are associated with diseases such as cancer (85). Several miRNAs have been implicated in inflammation and cancer through the activation of oncogenic and immune signaling pathways that converge in the expression of genes involved in activation of inflammation and carcinogenesis. Among these genes, the most important are miR-21, -125b, -155, -182, -196b and -210, which are known to be a source of immune and inflammatory stimuli (86).
The expression of miR-21, miR-155 and miR125b is controlled by an undetermined amount of immune signals, with TLR, TNF-α and cytokines being the most prominent signals that involve these miRNAs in inflammatory events (86). miR-155 expression is induced synergistically in NK cells following co-stimulation with IL-12 and IL-18 or only with IL-12. High levels of miR-155 expression enhance the induction and secretion of IFN-γ through the activation IL-12 and IL-18, whereas reduced miR-155 expression has the opposite effect (87). miR-21 is upregulated, in vitro and in vivo, by the oncogenes Ras or Src, the most frequently activated in human cancers (88). The mechanisms used by miRNAs to promote the initiation and progression of tumors include mechanisms that affect the modulation of by TLR, cytokines and their signaling pathways. Since cytokine signaling is essential for the differentiation of different cell subsets of the immune response, the miRNAs act as a limiting signal for differentiation these cells by modulating the expression of cytokines and their signaling components (86). miRNAs also play an important role in the development of cancers associated with infectious agents. Infection with various pathogenic agents induces changes in the expression of miRNAs functionally related to the mounting of the innate immune response, which regulates the survival and proliferation of immunocompetent cells and controls the infections. Regardless of whether the infectious agent is bacterial, viral or parasitic, the expression of miR-21, miR-125, miR-155 and miRNAs is the most commonly affected during infection and, therefore, they have a potential role in infection-induced carcinogenesis. It has been demonstrated that upregulation in the expression of miR-21 and miR-182 is associated with HR-HPV-associated carcinogenesis (14,86).

B lymphocytes mediate chronic inflammation
Using an HPV16-expressing transgenic mouse model, de Visser et al (89) analyzed the interaction between innate and adaptive immunity in the progression of the tumor induced by the virus. The authors created a lineage of HPV16-expressing mice, with an inability to produce B and T cells, by crossing a line transgenic for HPV16 with a lineage deficient for B and T cells. The genetic elimination of the adaptive immune response blocked the recruitment of the cells of the innate immune response, as well as blocking the remodeling of tissue, angiogenesis and carcinogenesis. When B cells obtained from transgenic HPV16-expressing animals were transferred to the T and B cell-deficient HPV16-expressing animals, the inflammatory response and cancer progression were restored, demonstrating that B cells are responsible for this phenomenon. When B cells are not infiltrates in precancerous tissues, it is hypothesized that they should remotely mobilize the cells of the innate immune response. This was confirmed by the finding that the serum-free cells obtained from transgenic animals also restored the progression of cancer in animals deficient in T and B cells (89).
The absence of B cells at the lesion site does not exclude the possibility of the involvement of antibodies directed against molecules expressed on the surface of tumor cells. Therefore, this phenomenon could be attributed to soluble mediators that promote interactions with APCs derived from the skin with the drainage of these cells to the lymph nodes and /or spleen where they would be able to trigger chronic inflammation. With regard to the type of soluble mediator produced by activated B cells at a distance that could be involved in the activation and maintenance of chronic inflammation in mice transgenic for HPV16, it has been reported that B cells produce chemokines including chemokine (C-X-C motif) ligand (CXCL) 1 and keratinocyte chemoattractant, in addition to cytokines IL-4 and IL-10 (90). These mediators have been implicated in the suppression of innate immunity during the assault by pathogens and/or in acute inflammation (91). Thus, inflammation could be involved in mediating the recruitment of leukocytes to neoplastic skin. The mechanisms of participation of these mediators entails the formation of an antigen-antibody complex, the activation of the complement and release of the anaphylatoxins C3a and C5a, which are potent proinflammatory factors. Alternatively, antibodies can activate the innate immune response through direct involvement by binding to multimeric Fc receptors present on the cell surface. Each case would lead to an inflammatory reaction (89). The genetic elimination of T and B cells does not increase the incidence of progression of premalignant lesions to HPV-associated carcinoma, on the contrary, it reduces the risk of this occurring, which suggests that adaptive immunity somehow participates in the process of chronic inflammation and carcinogenesis (89). This contradicts what has been observed in clinical and epidemiological studies, where it has been revealed that immunosuppressed humans are at higher risk for certain types of malignancies, including non-Hodgkin's lymphoma induced by Epstein-Barr virus and Kaposi's sarcoma associated with Kaposi's sarcoma-associated herpes virus (8,89). However, the relatively high incidence of these malignancies in immunocompromised patients may be at least partly explained by the greater susceptibility to infection and/or reactivation of latent infections caused by these viruses (8).

Chronic inflammation and HPV-induced cancer
Currently there is no more doubt that the persistent infection of the cervical mucosa with high-risk HPV (HR-HPV) is a necessary cause for the development of squamous intraepithelial neoplasia (SIL), low-grade SIL (LSIL) high-grade SIL (HSIL) and invasive cervical cancer (ICC). However, numerous women are infected with HR-HPV, but the majority of them never develop disease, suggesting that other factors must contribute to the development of the lesions and the transition from premalignant to malignant lesions. Prior to the discovery of the HPV, infection with C. trachomatis and HSV-2 were considered to be major risk factors for the development of ICC. However, it is now known that this association had probably been mistaken for a long time, due to a lack of methods able to detect the presence of HPV in tumor cells (91).
In past decades, studies have reported an association between chronic inflammation and the occurrence of HSIL and ICC (92)(93)(94). Furthermore, it was demonstrated that in infected females with HR-HPV, the presence of HSIL was highly associated with inflammation. This reinforces the concept that inflammation may be an important cofactor, contributing with HPV in the development of HSIL (95,96). Furthermore, was also demonstrated that seminal fluid contains a variety of active molecules, including cytokines, angiogenic factors, proteases, protein kinases, carrier proteins, structural proteins and molecules of the immune response, which are associated with inflammation (97). Considering that the development of HSIL and ICC requires an inflammatory environment, it has been proposed that these mediators present in seminal fluid could act to cause inflammation in the cervical mucosa, increasing the risk of lesions. Thus, it is possible that the deposition of seminal fluid in the mucosa of the female genital tract triggers a wave of cytokine release, with the recruitment and activation of leukocytes resulting in inflammation (98). This is consistent with previously reported data, which revealed that the use of condoms during sexual intercourse, despite not offering significant protection against genital infection by HPV, significantly reduces the occurrence of lesions in cervical mucosa caused by this virus (99). This mucosal protection offered by condoms against HPV infection, is likely due to them preventing contact between the semen and the epithelium of the cervical mucosa.
Notably, Schwebke and Zajackowski (93) hypothesized that inflammation of the uterine cervix, but not the diagnosis of a specific sexually transmitted disease, is associated with an increased risk of SIL and ICC. However, it is likely that the increased risk of cancer associated with chronic inflammation is due, at least in part, to the presence of infection with other sexually transmitted agents, including HSV-2, C. trachomatis and others, that cause an intense local inflammatory response (11,89). Various studies have demonstrated that the development of cancer induced by HR-HPV is preceded by chronic long-term inflammation, which in numerous cases, is developed with the participation of other sexually transmissible infectious agents, including HSV-2 and C. trachomatis (81,82,100,101).
Histological analysis of biopsies of cervical mucosa from HR-HPV-infected females revealed a higher degree of inflammation, with an increased infiltration of lymphocytes and neutrophils into the epithelium, compared with those who were uninfected. The association between persistent infection with a specific HPV type and progression of LSIL to HSIL was less evident in women with moderate inflammation in the stroma or in the epithelium. In individuals infected with HR-HPV, the inflammation tended to involve only the surface layer of the epithelium, instead of the basal layer, or involvement extended to all the cervical epithelium (96). This suggests that inflammation varies with HPV type, persistent or not, and possesses a risk of progression.
Assessing the number of macrophages present in the cervical epithelium of healthy females and those with LSIL, HSIL and ICC, Hammes et al (96) identified a positive linear association between the amount of macrophages and the progression of lesions, finding that an increased migration of macrophages to the epithelium was proportional to the worsening of the injury. A direct association between the amount of macrophages present in the epithelium and the progression of premalignant lesions to cancer was also identified. The intensity of inflammation was also closely associated with the degree of the lesion. The macrophage migration from the epithelium to the stroma was influenced by not only inflammation, but also by the presence of dysplastic cells. These data reveal that cluster of differentiation (CD)68 + macrophages are associated with cervical carcinogenesis, promoting the progression of intraepithelial lesions to invasive forms.
The role of the E6 and E7 HPV16 oncogenes in the regulation of IL-1β expression was analyzed by Niebler et al (102) in HPV16-immortalized human keratinocytes and in cells obtained from cervical cancer. These researchers found that in keratinocytes expressing only E7, the secretion of IL-1β was highly inducible by the activation of inflammation; however, in those expressing only E6, no production of IL-1β was detected and the IL-1β precursor and p53 were degraded in a proteasome-dependent manner, mediated by ubiquitin ligase E6-AP. By contrast, in keratinocytes expressing E6 and E7 or only E7, the levels of the IL-1β precursor were restored by interference RNAs, which block the expression of E6-AP, with simultaneous recovery of functional p53. The results from this study indicated the presence of a novel mechanism for post-translational regulation of the IL-1β precursor that inhibits IL-1β secretion in HPV-infected keratinocytes. This reveals an effective and innovative mechanism of HR-HPV to prevent the function of IL-1β, which reduces the innate immune response against the infected cells, facilitating persistence of the virus, which is an important step in the initiation of the cell transformation and tumorigenesis (102).
The systemic levels of proinflammatory cytokines in female patients with persistent HPV infection were evaluated by Kemp et al (103), who found significantly elevated levels of IL-6, IL-8, TNF-α, macrophage inflammatory protein (MIP)-1α, granulocyte-macrophage colony-stimulating factor, IL-1α and IL-1β in the plasma of the patients with the infection compared with uninfected individuals. It was also noted that high systemic levels of these cytokines were accompanied by a reduction in lymphoproliferative response. This is a novel finding and is somewhat unexpected as there is little evidence of an HPV-induced systemic inflammatory reaction. The presence of proinflammatory cytokines in the peripheral blood was associated with HPV persistence and weak lymphoproliferative response (103).
Analyzing the profile of immune response in a case-control study, Peghini et al (13), found that 75% of individuals with normal cytology exhibited a profile of Th1 cytokines, compared with only 10% of women with LSIL, HSIL or ICC. The samples of ICC exhibited more commonly a Treg profile and behaved in the opposite way to Th1. Changes in the profile of cytokines present in the microenvironment were also revealed during the progression of lesions, demonstrating a significant reduction of IL-2, IL-12 and TNFα, and an increase in TGFβ with aggravation of the lesions. The pattern of cytokines found during tumor progression makes it clear that there is involvement of Treg cells in the induction of ICC. Tumor progression was dependent on the suppression of cellular immunity since normal individuals or individuals with less severe lesions were observed to possess a profile of Th1 cytokines, with a change to an immunosuppressive Treg profile in those with neoplastic progression (13). This suggests that a microenvironment with chronic inflammation and an imbalance in the Th1/Treg ratio and the levels of cytokines produced appears to be a critical mechanism for tumor-cell evasion of the immune surveillance system, facilitating tumor progression.
The exact mechanism of the clearance of HPV infection remains unclear. HPV persistence is considered to require a tolerant local immune environment, favoring evasion of the virus or the suppression of the innate and adaptive immune responses (103). The absence of viremia and cytolysis in cervical HPV infection contributes to the difficulty in defining the immunological mechanisms regulating the clearance of the virus (76,104). It is thought that the innate immune response has a critical role early in the process of healing of HPV infection (105).
Analysis of the signaling of PRRs in uninfected and newly infected keratinocytes, and those persistently infected with HR-HPV, revealed that active infection impairs emission of signals from PRRs to the cell nucleus, affecting the production of type 1 IFNs, proinflammatory cytokines and chemokines. This mechanism of suppression is dependent on the expression of the intracellular protein ubiquitin carboxyl-terminal L1 (UCHL1), which is induced by HR-HPV in keratinocytes. UCHL1 mediates the degradation of NF-κB and promotes phosphorylation of p65, resulting in the chronic suppression of NF-κB signaling, an important modulator of cellular immunity. This indicates that HR-HPV uses UCHL1 to escape the innate immunity of the host, by suppressing the production of interferons, cytokines and chemokines induced by PRRs, which are necessary for the attraction and activation of cells that are also involved in the adaptive immune response (106).
Notably, following the integration of HR-HPV into the genome of the host cell, NF-κB appears to go on to have an important role in the development of ICC, since this transcription factor was detected in nucleus and cytoplasm of 96.4% of HPV-positive tumor samples and in only 52.9% of HPV-negative tumor samples. The overexpression of NF-κB in ICC suggests that NF-κB exerts modulating effects on chronic inflammation and increases the risk of cancer. The mechanism of action of NF-κB in the process of HPV-induced cervical carcinogenesis is probably due to the action of the viral oncoproteins E6 and E7 that, besides increasing the transcriptional activity of NF-κB, eliminate the functions of cellular proteins p53 and pRB, leading to an uncontrolled cell cycle with induction of immortalization and neoplastic transformation of the cell (107). It has been demonstrated that E7 induces expression of IL-1β which, in turn, activates the expression of NF-κB, while E6 is associated with the nuclear localization of this transcription factor. Thus E6 and E7 are associated with the increased activity of NF-κB that is a key modulator in the transition from chronic inflammation to cancer (108,109).
In a recent multiethnic cohort study, Scott et al (110) monitored females between 2005 and 2010 to evaluate the expression levels of candidate antiviral cytokines in mucosa, including IFN-α2, IFN-γ, IL-12 and IL-10, as well as the proinflammatory cytokines (IL-1α, -1β, -6 and -8, CXCL8, MIP-1α, CCl3 and TNF-α) that are induced following the establishment of HPV infection. The majority of the cohort exhibited increased expression of certain cytokines and decreased expression of others following infection. An association was observed between high levels of IL-10, IL-12, MIP-1α and TNF-α in the cervical mucosa and a reduced likelihood of clearance of any HPV type. For HR-HPV, the trend of no viral clearance was significantly higher in the presence of high levels of IL-12 and TNF-α. Similar patterns were observed in infection with low-risk HPV, although the trend of not healing was more modest, revealing a significant difference only for IL-10. In the group of women who had rapid healing of HPV infection, low levels of cytokines were associated with a more rapid clearance of the virus, compared with the group of women with established long-term infection. The clearance of HR-HPV was more frequent in the presence of low levels of MIP-1α and IL-8 expression in the cervical mucosa in the group of women that demonstrated rapid healing of infection, but not in the group with established long-term infection. Increased local levels of several important proinflammatory cytokines, including TNFα, MIP-1α, IL-12 and IL-10, were associated with a decreased likelihood of elimination of the virus in women with established long-term infection (110). Scott et al made two considerations regarding the results. The first is that in individuals with transient exposure to the virus, in the absence of productive infection, the induction of cytokines may not occur and the virus can be eliminated by a non-immune mechanism. This would explain the absence of high levels of cytokines among women with rapid clearance of the virus. The second consideration is that, in a successful immune response, the immune mechanisms of homeostatic control may precede the elimination of the virus, bringing the previously high cytokine levels back to baseline levels in anticipation of the elimination of the virus.
There is evidence to support a significant role of TNFα in the immune response against HPV infected cells, as well as in the natural history of disease associated with this virus, since expression of this cytokine by infiltrating mononuclear cells is correlated with spontaneous regression of the lesions induced by HPV. Furthermore, it has been demonstrated that the HPV E6 and E7 proteins suppress the protective effect of TNFα, as E6 induces resistance to TNFα-mediated apoptosis (110) and E7 inhibits the antiproliferative effect of this cytokine (109). This suggests that acquisition of resistance to TNFα may be an important step in HPV-induced carcinogenesis. However, there is also evidence that, under certain conditions, TNFα can act as a tumor promoter (111). This is an eloquent demonstration of the complexity of the interactions established between the broad spectrum of cytokines produced during the inflammatory response, in which certain cytokines have effects and functions that are occasionally contradictory, depending on the context in which they operate.
Persistent infection with HR-HPV results in the integration of viral DNA into the host cell genome and overexpression of oncogenes E6 and E7, which are considered key factors for the development of ICC. These viral genes encode the E6 and E7 oncoproteins, which are responsible for the transforming activity of infected cells. The main targets of these viral proteins are the cellular tumor suppressor proteins p53 and pRB, which are responsible for cell cycle control (112)(113)(114). Though oncogenes E6 and E7 are major HPV genes involved in neoplastic transformation, evidence points to an important role of the E5 viral oncogene in tumorigenesis and in modulation of immune cells (115). E5 is also involved in the regulation of late viral functions, together with viral gene E4. The E1 and E2 viral genes encode viral replication factors and appear to play a role in HPV persistence, enabling copies of the virus to be maintained in an episomal form in the nucleus and be transferred to daughter cells during mitosis (73,116). Within the epithelial cells, the HPV E6 protein binds to the cellular protein p53 and promotes its degradation by an ubiquitin-dependent pathway, while the HPV E7 protein binds to the pRB family members, consisting of p105, p107 and p130, and promotes their degradation. The elimination of the functions of p53 and pRB results in uncontrolled cell cycle progression and loss of DNA repair mechanisms, with the consequent accumulation of mutations creating genomic instability (72,73). Furthermore, the expression of HPV E6 and E7 genes in differentiating keratinocytes directly alters the expression of genes that influence host resistance to infection and immune functions. A primary function of E6 and E7 is to maintain the keratinocytes in the cell cycle, but they also prevent antiviral and antitumor effects, and exert immunoregulatory action of the IFN-mediated innate immune system. E7 protein blocks IFN-α activity by inhibiting the expression of the inducible genes that lead to IFN-α production. E7 also inhibits the production of IFN-β by inhibiting the activation of the IFN-β promoter (113). The changes in cellular functions triggered by the action of viral oncoproteins can also result in increased production of nitric oxide, DNA damage, and activation of cyclooxygenase-2/prostaglandin/prostaglandin receptors (COX2/PGE/PGERs), an inflammatory pathway that leads to increased inflammation and tumorigenesis. Inflammatory and tumor cells can then release cytokines, chemokines and prostaglandins which act in an autocrine/paracrine manner to regulate the function of endothelial, stromal, neoplastic, epithelial and infiltrating immune cells to cause increased tumor angiogenesis, increased tumor growth, decreased apoptosis and decreased local immune-surveillance. These conditions favor tumorigenesis and permanence of the virus in the tumor microenvironment. Inhibition of the COX-PGE cascade with nonsteroidal anti-inflammatory drugs such as aspirin could reduce inflammation and tumor progression (82,117).
The relationship between inflammation, angiogenesis and HPV-induced lesions was assessed by Mazibranda et al (12), in samples obtained from patients with normal cytology, CIN and ICC. Based on the density of pan-endothelial cells present on-site, identified by the CD31 marker, a progressive increase in microvessels was observed, with increasing severity of the lesion. Macrophage infiltration was associated with neovascularity in CIN and ICC, with a strong positive correlation between the number of infiltrating macrophages in the tumor and its vasculature. An increase in macrophages in microvessels was also observed in parallel with neoplastic progression, being even higher in ICC (12).
Immune evasion is an essential aspect of HPV persistence and is also essential for the development of ICC. As there is no sign of viremia, cytolysis or necrosis in the early-phase HPV-infected cervical epithelium, even when it is productive, there is not an adequate activation of the innate immune system or inflammation. This is due to the fact that the viral E6 and E7 oncoproteins inhibit the expression of PRRs, particularly TLR9, promoting dysregulation of the interferon signaling pathway, preventing activation of the innate immune response and allowing cells to remain infected with HPV, which uses the cellular machinery for viral replication followed by persistence (109,112). Once established, persistent HPV infection leads to changes in the release of proinflammatory cytokines, which in turn may alter the infiltration of immune cells, causing inflammation. Changes in immune response and elevated systemic levels of proinflammatory cytokines have been observed in older women, ~50 years old, with persistent HPV infection (111). It was demonstrated that in neoplastic epithelial cells of the cervical mucosa, the E5, E6 and E7 HPV16 oncogenes were able to induce the cyclooxygenase (COX)-prostaglandin inflammatory axis, increasing the immediate expression of the COX2 gene (82). These results suggest a direct link between the HPV oncogenes and activation of potent inflammatory cascades with well-known roles in cancer promotion. Thus, although HPV is not associated with inflammation at the initiation of infection, it is likely that, following its integration, persistence and transformation leads to the activation of inflammatory pathways, including the COX-prostaglandin pathway, in neoplastic epithelial cells, promoting immune cell infiltration, inflammation, and tumor progression (118). Biopsy studies of tumors associated with HPV reveal significantly higher expression levels of COX1 and COX2 in neoplastic epithelial cells and vascular endothelial cells of all grades and stages in ICC (110,109). These findings suggest an important role for the two isoforms of COX in the pathology of disease. Furthermore, it was revealed that the induction of COX1 expression in HeLa cells, a lineage positive for HPV18, caused a rapid and sustained elevation of COX2 and prostaglandin synthase terminal, resulting in the synthesis of PGE2 (109). Furthermore, PGE2 was produced by COX1 and COX2, indicating that they may contribute equally, or act synergistically, to promote ICC (118).
The mechanism for the selective production of prostaglandin is determined by terminal prostaglandin synthase, which depends on the PTGES synthase enzyme present in COX1-and COX2-expressing cells that converts prostaglandin H2 into PGE2 (98). It was demonstrated that this enzyme is represented significantly in ICC, and that products of the oncogenes of HPV and PGE2 can regulate the expression of the prostaglandin E receptor PTGER (119).
Furthermore, it was demonstrated that the E5 protein of HPV16 regulates the expression of PTGER4 in the cells obtained from ICC, so that the production of PGE2 is cyclic adenosine monophosphate (cAMP) dependent (115). These results suggest that increased levels of PGE2 in ICC can regulate the function of neoplastic cells in an autocrine or paracrine manner, through the expression of high levels of the prostaglandin receptors PTGER2 and PTGER4.
There is evidence to indicate that the initial HPV infection is followed by integration of the viral DNA into the genome of the epithelial cells of the cervical mucosa and activation of viral oncogenes, leading to the induction of COX1 and 2 expression, PTGE synthase expression, PGE2 biosynthesis and PTGER expression. In turn, PGE2 can regulate the function of tumor cells through PTGER by the cAMP signaling pathway (118). Certain immunological parameters were evaluated in women infected with HR-HPV, without lesions, CIN1, CIN3, carcinoma in situ (CIS) and ICC. The results revealed that the cervical mucosa was infiltrated by B cells expressing the CD20 and CD138 markers as early as in CIN1 and this infiltration increased with the worsening of the lesion, being strongly correlated with the infiltration of lymphoid cells expressing the marker CD32B and lymphocytes expressing FoxP3. The lymphoid cells GATA3 and T-bet were found in greater amounts in samples from patients with ICC compared with those obtained from women with normal cytology. The expression levels of the cytokines thymic stromal lymphopoietin and indoleamine 2,3-dioxygenase-1, promoting Th2 response and serum levels of IL-10, in epithelial cells were higher in the samples from patients with CIN3/CIS and in ICC compared with cytologically-normal samples. An increased infiltration of CD138-, CD20-or CD32B-expressing lymphoid cells into the stroma were observed as early as in CIN1. This indicates that a Th2-type response is present from the first histological evidence of pre-neoplastic transformation caused by HR-HPV. Furthermore, the presence of stromal lymphocytes expressing FoxP3, a marker expressed primarily by Treg cells, was detected in CIN3/CIS and ICC (120).
In a case-control study, Chen et al (33) analyzed the cytokine profile and calculated the percentages of Treg and Th17 cells in the peripheral blood of females with normal cytology, CIN1 and ICC. They observed a significant increase in the levels of TGF-β, IL-6, -10, -17 and -23 expression, and lower expression levels of IFN-γ, in patients with CIN1 and ICC compared with those with normal cytology. These results suggest that the immune response in patients with ICC has been suppressed. It was also observed that the Th17/Treg ratio in the patients with CIN1 and in those with ICC was significantly higher than in the control group. These data suggest that in patients with ICC, the percentage of Th17 and the Th17/Treg ratio were significantly altered. Also, a positive correlation was found between the percentages of Th17 and expression levels of IL-6, -23 and -17, all of which were higher in patients with ICC. A positive correlation was also found between the percentages of Treg and expression levels of TGF-β and IL-6. By contrast, there was a negative correlation between the percentage of Treg cells and the expression levels of IFN-γ (33). These data indicate an imbalance in the TH17/Treg ratio in the peripheral blood of patients with ICC. In a previous study, Zhang et al (32) had already found the percentage of circulating Th17 and Treg cells to be significantly higher in patients with CIN and ICC compared with a healthy control group. This study also reported an important imbalance in the average Th17/Treg ratio in patients with CIN or ICC. Furthermore, IL-17 and IL-10 levels were significantly higher in patients with ICC compared with the control group. The levels of Th17 and Treg cells gradually increased during the progression of the disease, leading to an imbalance of the TH17/Treg ratio in patients with ICC, suggesting a potential role of the Th17/Treg imbalance in the progression of CIN to cancer.

Conclusion
Long-term chronic inflammation in addition to persistent HPV infection is one potential cause for the development of ICC, involving the participation of reactive species of oxygen and nitrogen, cytokines, chemokines, growth and survival cell factors, enzymes including COX and metalloproteinase, prostaglandins and specific types of microRNAs. The collective action of these mediators induces changes in the processes of proliferation, senescence and cell death, and also in mutation, methylation of DNA and angiogenesis, contributing to development of HPV-induced ICC. Persistent infection with HR-HPV leads to the integration of the viral DNA into the genome of the host cell, with the overexpression of the viral oncogenes E5, E6 and E7. The product of E5 promotes fusion between cells, causing aneuploidy, and favors the transforming activity of E6 and E7 and immune evasion of the virus. In addition, E5 reduces the expression of class I major histocompatibility complex and causes inflammation by increasing COX2 expression which, in turn, increases the secretion of prostaglandin and its receptors. The products of the E6 and E7 genes eliminates the function of p53 and intracellular pRB, resulting in a loss of control of the cell cycle and of DNA repair, favoring accumulation of mutations and increasing the risk of neoplastic transformation. E6 activates telomerase, preventing senescence and cell death, leading to immortalization of the cell. E7 induces the expression of IL1β, which, in turn activates the expression of NF-κB, a key modulator in the transition of chronic inflammation to cancer. E6 and E7 remain correlated with production of the matrix metalloproteinase enzyme, which acts in remodeling the vascular system, facilitating diapedesis and angiogenesis. The high levels of TGF-β and IL-6, -10, -17 and -23 and the low level of IFN-γ in patients with ICC compared with healthy females, suggesting that the immune response was suppressed in ICC patients. Also, the percentage of Th17 and the TH17/Treg ratio are significantly altered, which suggests that the microenvironment of chronic inflammation changes the cytokine profile and promotes an imbalance in Th1/Treg, that appears to be critical to tumor-cell evasion of the immune surveillance system. ICC favors cellular proliferation, inhibits apoptosis and promotes tissue remodeling and angiogenesis, increasing the risk of tumor progression and metastasis.


extfieldsofstudy: Biology Medicine

text:
The Essential Role of Cytokinin Signaling in Root Apical Meristem Formation during Somatic Embryogenesis

Plant tissue culture and subsequent organogenesis have been regarded as part of the scientific roots of modern plant biotechnology (Sussex, 2008). Establishments of shoot apical meristem (SAM) and root apical meristem (RAM) are critical steps for somatic embryogenesis (Scheres, 2007). WUSCHEL (WUS), a homeodomain protein, controls stem cell differentiation and maintenance and its ectopic expression facilitated the transition from vegetative growth to embryogenesis in Arabidopsis (Zuo et al., 2002). Gain-of-function of WUS mutant can trigger cell pluripotence and re-establishing new meristems in the cortex, which further reinforced the essential role of WUS in the initiation and maintenance of meristem in planta (Xu et al., 2005). Nevertheless, the underlying mechanisms of how somatic cell maintains plasticity and triggers cellular switch during the sex-free embryogenesis are still elusive, which is one of the top 25 profound as-yet-unanswered scientific questions (Vogel, 2005). During early somatic embryogenesis, while auxin signaling cascade induces the expression of WUS (Su et al., 2011), little is known about the underlying mechanisms for the RAM establishment.

The quiescent center (QC) is formed from apical descendant cell after asymmetric division of the hypophysis and essential in maintaining the identity of root stem cells (Moller and Weijers, 2009; Peris et al., 2010). WUSCHEL-RELATED HOMEOBOX 5 (WOX5) plays an analogous role in the QC of the RAM as WUS in organizing center (OC) of the SAM. PLETHORA1 (PLT1) and PLT2, two AP2-type transcription factors, are required to specify and maintain the stem–cell identity in the RAM (Galinha et al., 2007). In addition, transcription factors SCARECROW (SCR) and SHORTROOT (SHR) are necessary to maintain stem cell activity in the RAM (Sabatini et al., 2003). In this study, Dr Xian Sheng Zhang's lab exploited the marker lines of the above genes to investigate the specification and initiation of RAM during nongametic somatic embryogenesis. They detected almost simultaneously induced WOX5 and PLT2 expression in some regions of embryonic callus only 24 h after transferred onto 2,4-D free somatic embryo-inducing medium (SEIM), while the expression of SCR was detected later in 24–36 h. Further, the spatiotemporal expression patterns of WUS and WOX5 were examined by tagged with distinct fluorescence protein markers. Nearly identical expression patterns of them were found after 24 h induction. The WOX5 signals were then detected below the regions of WUS signals, suggesting that an early shoot-root axis had been established at this time. Su and colleagues also employed inducible antisense WOX5 transgenic lines and plt2 mutant to investigate their roles for embryonic root formation. Strikingly, significantly decreased number of somatic embryos (SEs) and severely defected SEs were produced from embryonic callus of WOX5 antisense lines and plt2-1 mutant respectively, confirmed that WOX5 and PLT2 are required for somatic embryogenesis, and further indicated the role of WOX5 in the RAM is comparable to that of WUS in the SAM.

In zygotic embryos, auxin and cytokinin display inverse correlation in specifying the root stem cell niche (Muller and Sheen, 2008). In this scenario, auxin signaling antagonizes cytokinin responses during zygotic embryogenesis by directly inducing the transcription of ARABIDOPSIS RESPONSE REGULATOR (ARR) 7 and ARR15, the two critical A-type ARR transcription factors to repress cytokinin signaling (Figure (Figure1),1), which is essential in establishing the root stem cell niche (Muller and Sheen, 2008). Intriguingly, ARR7 and ARR15 expression is stimulated during root specification, but repressed by the auxin signal in the SAM of the embryo (Zhao et al., 2010). However, the interaction of auxin and cytokinin in the establishment of root stem cell niche in somatic embryogenesis is less clear. Su and her colleagues further traced auxin response signals by utilizing DR5rev promoter driven VENUS or GFP expression and found that its spatiotemporal expression patterns were overlapped with neither WOX5:GFP nor PLT2:RFP. Instead, they showed that ARR7 and ARR15, which also can be induced by cytokinin (Werner and Schmulling, 2009), displayed overlapping expression patterns with WOX5 rather than WUS. This remarkable evidence implied that cytokinin, but not auxin, is essential for initiation of the RAM of SE (Figure (Figure1).1). This was further supported by the observation of defective SEs in the overexpressing lines of ARR7 and ARR15, and double mutants of ahk2ahk4 and ahk3ahk4. The authors finally demonstrated that cytokinin signaling strictly confined WOX5 expression to the site of subsequently embryonic root meristem by in situ hybridization (Su et al., 2015).

Figure 1

Comparison of the roles of auxin and cytokinin during RAM specification in zygotic and somatic embryos. The cells with green color represent established RAM niche in zygotic embryogenesis (ZE) and somatic embryogenesis (SE) respectively. The dashed lines ...

The establishment of the shoot-root axis is a crucial process for both zygotic and somatic embryogenesis. Despite the processes of the two kinds of embryogenesis are quite similar from globular to torpedo stage (Zimmerman, 1993), the underlying mechanisms seem to be different in at least two aspects. (1) The spatiotemporal expression patterns of WUS and WOX genes are distinct. In zygotic embryogenesis, WUS and WOX5 are sequentially expressed with roughly 12 h interval. While during somatic embryogenesis they are simultaneously expressed in nearly overlapping regions of embryonic callus, indicating that SAM and RAM are almost initiated at the overlapping regional domains of callus and formed the apical-basal axis at early stages of somatic embryogenesis. (2) The roles of auxin gradient response and cytokinin signaling show discrepancy in zygotic and somatic embryogenesis (Figure (Figure1).1). In planta, the proper establishment of auxin gradient response is essential for correct spatiotemporal cytokinin signaling, which causes the lower expression of ARR7/ARR15 in the upper lens-shaped cell but higher expression in the lower large basal cell (Muller and Sheen, 2008). Cytokinin signaling plays a critical role during root stem cell niche formation in zygotic embryogenesis as chemical induced double mutant arr7arr15 produced the defective RAM system. In here, Su and her colleagues' compelling results demonstrated that cytokinin signaling is also essential for RAM initiation in SE. Collectively, their results provide novel insights into the roles of cytokinin and auxin in the formation of shoot-root axis during somatic embryogenesis, and contribute to the ongoing investigation of the mechanisms of somatic embryogenesis. These knowledges are invaluable in propagating the endangered and indispensable plant species and in molecular breeding of crops through callus-based plant transformation in the near future.

Plant tissue culture and subsequent organogenesis have been regarded as part of the scientific roots of modern plant biotechnology (Sussex, 2008). Establishments of shoot apical meristem (SAM) and root apical meristem (RAM) are critical steps for somatic embryogenesis (Scheres, 2007). WUSCHEL (WUS), a homeodomain protein, controls stem cell differentiation and maintenance and its ectopic expression facilitated the transition from vegetative growth to embryogenesis in Arabidopsis (Zuo et al., 2002). Gain-of-function of WUS mutant can trigger cell pluripotence and re-establishing new meristems in the cortex, which further reinforced the essential role of WUS in the initiation and maintenance of meristem in planta (Xu et al., 2005). Nevertheless, the underlying mechanisms of how somatic cell maintains plasticity and triggers cellular switch during the sex-free embryogenesis are still elusive, which is one of the top 25 profound as-yet-unanswered scientific questions (Vogel, 2005). During early somatic embryogenesis, while auxin signaling cascade induces the expression of WUS (Su et al., 2011), little is known about the underlying mechanisms for the RAM establishment.
The quiescent center (QC) is formed from apical descendant cell after asymmetric division of the hypophysis and essential in maintaining the identity of root stem cells (Möller and Weijers, 2009;Peris et al., 2010). WUSCHEL-RELATED HOMEOBOX 5 (WOX5) plays an analogous role in the QC of the RAM as WUS in organizing center (OC) of the SAM. PLETHORA1 (PLT1) and PLT2, two AP2-type transcription factors, are required to specify and maintain the stem-cell identity in the RAM (Galinha et al., 2007). In addition, transcription factors SCARECROW (SCR) and SHORTROOT (SHR) are necessary to maintain stem cell activity in the RAM (Sabatini et al., 2003). In this study, Dr Xian Sheng Zhang's lab exploited the marker lines of the above genes to investigate the specification and initiation of RAM during nongametic somatic embryogenesis. They detected almost simultaneously induced WOX5 and PLT2 expression in some regions of embryonic callus only 24 h after transferred onto 2,4-D free somatic embryo-inducing medium (SEIM), while the expression of SCR was detected later in 24-36 h. Further, the spatiotemporal expression patterns of WUS and WOX5 were examined by tagged with distinct fluorescence protein markers. Nearly identical expression patterns of them were found after 24 h induction. The WOX5 signals were then detected below the regions of WUS signals, suggesting that an early shoot-root axis had been established at this time. Su and colleagues also employed inducible antisense WOX5 transgenic lines and plt2 mutant to investigate their roles for embryonic root formation. Strikingly, significantly decreased number of somatic embryos (SEs) and severely defected SEs were produced from embryonic callus of WOX5 antisense lines and plt2-1 mutant respectively, confirmed that WOX5 and PLT2 are required for somatic embryogenesis, and further indicated the role of WOX5 in the RAM is comparable to that of WUS in the SAM.
In zygotic embryos, auxin and cytokinin display inverse correlation in specifying the root stem cell niche (Müller and Sheen, 2008). In this scenario, auxin signaling antagonizes cytokinin responses during zygotic embryogenesis by directly inducing the transcription of ARABIDOPSIS RESPONSE REGULATOR (ARR) 7 and ARR15, the two critical A-type ARR transcription factors to repress cytokinin signaling (Figure 1), which is essential in establishing the root stem cell niche (Müller and Sheen, 2008). Intriguingly, ARR7 and ARR15 expression is stimulated during root specification, but repressed by the auxin signal in the SAM of the embryo (Zhao et al., 2010). However, the interaction of auxin and cytokinin in the establishment of root stem cell niche in somatic embryogenesis is less clear. Su and her colleagues further traced auxin response signals by utilizing DR5rev promoter driven VENUS or GFP expression and found that its spatiotemporal expression patterns were overlapped with neither WOX5:GFP nor PLT2:RFP. Instead, they showed that ARR7 and ARR15, which also can be induced by cytokinin (Werner and Schmülling, 2009), displayed overlapping expression patterns with WOX5 rather than WUS. This remarkable evidence implied that cytokinin, but not auxin, is essential for initiation of the RAM of SE (Figure 1). This was further supported by the observation of defective SEs in the overexpressing lines of ARR7 and ARR15, and double mutants of ahk2ahk4 and ahk3ahk4. The authors finally demonstrated that cytokinin signaling strictly confined WOX5 expression to the site of subsequently embryonic root meristem by in situ hybridization (Su et al., 2015).
The establishment of the shoot-root axis is a crucial process for both zygotic and somatic embryogenesis. Despite the processes of the two kinds of embryogenesis are quite similar from globular to torpedo stage (Zimmerman, 1993), the underlying mechanisms seem to be different in at least two aspects. (1) The spatiotemporal expression patterns of WUS and WOX genes are distinct. In zygotic embryogenesis, WUS and WOX5 are sequentially expressed with roughly 12 h interval. While during somatic embryogenesis they are simultaneously expressed in nearly overlapping regions of embryonic callus, indicating that SAM and RAM are almost initiated at the overlapping regional domains of callus and formed the apicalbasal axis at early stages of somatic embryogenesis. (2) The roles of auxin gradient response and cytokinin signaling show discrepancy in zygotic and somatic embryogenesis (Figure 1). In planta, the proper establishment of auxin gradient response is essential for correct spatiotemporal cytokinin signaling, which causes the lower expression of ARR7/ARR15 in the upper lens-shaped cell but higher expression in the lower large basal cell (Müller and Sheen, 2008). Cytokinin signaling plays a critical role during root stem cell niche formation in zygotic embryogenesis as chemical induced double mutant arr7arr15 produced the defective RAM system. In here, Su and her colleagues' compelling results demonstrated that cytokinin signaling is also essential for RAM initiation in SE. Collectively, their results provide novel insights into the roles of cytokinin and auxin in the formation of shoot-root axis during somatic embryogenesis, and contribute to the ongoing investigation of the mechanisms of somatic embryogenesis. These knowledges are invaluable in propagating the endangered and indispensable plant species and in molecular breeding of crops through callus-based plant transformation in the near future.

AUTHOR CONTRIBUTIONS
LW and KC conceived the research and wrote the manuscript.