Picrasma quassioides Extract Elevates the Cervical Cancer Cell Apoptosis Through ROS-Mitochondrial Axis Activated p38 MAPK Signaling Pathway

Background/Aim: Picrasma quassioides (P. quassioides) is used in traditional Asian medicine widely for the treatment of anemopyretic cold, eczema, nausea, loss of appetite, diabetes mellitus, hypertension etc. In this study we aimed to understand the effect of P. quassioides ethanol extract on SiHa cervical cancer cell apoptosis. Materials and Methods: The P. quassioides extract-induced apoptosis was analyzed using the MTT assay, fluorescence microscopy, flow cytometry and western blotting. Results: P. quassioides extract induced cellular apoptosis by increasing the accumulation of cellular and mitochondrial reactive oxygen species (ROS) levels and inhibiting ATP synthesis. Pretreatment with N-Acetylcysteine (NAC), a classic antioxidant, decreased the intracellular ROS production and inhibited apoptosis. In addition, the P38 MAPK signaling pathway is a key in the apoptosis of SiHa cells induced by the P. quassioides extract. Conclusion: The P. quassioides extract exerts its anti-cancer properties on SiHa cells through ROS-mitochondria axis and P38 MAPK signaling. Our data provide a new insight for P. quassioides as a therapeutic strategy for cervical cancer treatment.

) are the principal active components in P. quassioides. These active materials show anti-inflammatory, antihypertensive and anti-angiogenic properties in humans (8,9). At the same time, recent studies have shown that the monomers extracted from P. quassioides have the ability to increase intracellular reactive oxygen species and induce apoptosis in HepG2 and Hep3B liver cancer cells (16,17). Despite these data, the anti-cancer effect of P. quassioides on cervical cancer and the possible underlying mechanism has not yet been studied.
Reactive oxygen species (ROS) formed from various cellular metabolites play a vital role in many biological processes, including oxygen sensing, cell differentiation and adaptive immunity (18). Disturbances in the balance between intracellular antioxidants and ROS can lead to the dysfunction of mitochondria (19), which can promote cell and tissue oxidative stress in vivo and in vitro. Most active radicals are considered as natural by-products of mitochondrial activation. Generally, low levels of ROS can serve as signal molecules to regulate numerous physiological processes, while excess levels can lead to caspase activation and cytochrome c release and promote cell death (20), causing various diseases, such as cardiovascular disease, diabetes, and cancer (21,22). Compared to normal cells, cancer cells have higher ROS levels. High levels of ROS change the redox state of the cell itself, promoting the occurrence and development of malignant phenotypes as reflected in the continuous proliferation of cancer cells (20).
The biochemical characteristics of cancer cells make them more vulnerable to further ROS increase induced by exogenous ROS-generating agents (pro-oxidants) compared to normal cells (23). Based on these facts, new anti-cancer strategies for ROS have been developed to selectively kill cancer cells by promoting excessive ROS production and inducing cancer cell mitochondrial dysfunction (24)(25)(26).
Herein, the effect of P. quassioides extraction on the survival of cervical cancer was evaluated by examining the viability, apoptosis and the changes in apoptosis-related protein expression in SiHa cervical cancer cells. We also investigated the effect of P. quassioides extract on SiHa cell migration, colony formation, mitochondrial membrane potential, cellular and mitochondrial ROS levels and ROSdependent signaling pathways, such as MAPK signaling. Our findings may give a new sight in understanding the potential anti-cancer properties of P. quassioides extract on cervical cancer.

Materials and Methods
Extract preparation. A crude extract of Picrasma quassioides was prepared essentially as described before (27). Briefly, air-dried stems of Picrasma quassioides (50 g) were soaked in 500 ml of ethyl alcohol, and then extracted by shaking it at 200 rpm for 5 h at 37˚C. The filtrate was recovered, and an additional 500 ml of ethyl alcohol were used for filtering the residue. The process of recovery and filtration of the extract was repeated three times and it was concentrated on a rotary evaporator for 12 h at 55˚C. The concentrated extract was labeled as PQ and stored in a refrigerator at 4˚C until further use.

Apoptosis detection by
Colony-formation assay. SiHa cells (1×10 3 cells/well) were seeded in six-well plates, treated with PQ (60μg/ml) and maintained at 37˚C in a 5% CO 2 incubator for 10 days. The cells were then washed with 1×PBS, fixed for 10 min with 3.7% formaldehyde, treated for 20 min with methanol, and stained for 30 min with 0.05% crystal violet. The plates were washed with 1×PBS three times before image capturing.
Detection of ROS production. Cells were seeded in 6-well plates at 2×10 5 cells/well and treated with PQ for 24 h. The changes in cellular and mitochondrial ROS levels were determined using Dihydroethidium (DHE. Beyotime Biotechnology, Shanghai, P.R. China), which is specific for superoxide and hydrogen peroxide in living cells and MitoSOX (Thermo Fisher Scientific, Waltham, MA, USA) staining, which detects superoxide in the mitochondria of living cells. Nuclei were visualized using the Hoechst 32258 (Thermo Fisher Scientific, Waltham, MA, USA) staining. Nuclear staining was observed qualitatively under a microscope after 20 min of incubation with the Hoechst dye.  were determined using JC-1 (Beyotime, Shanghai, PR China). Cells were incubated with 20 mM JC-1 for 15 min at 37˚C and then washed with PBS. After washing with PBS, images were taken using a fluorescence core cell culture microscope (EVOS ® x) and the fluorescence intensities were observed qualitatively.

. Effect of PQ on cellular and mitochondrial ROS, mitochondrial membrane permeability and ATP content in SiHa cells. (A) Dihydroethidium (DHE) and Hoechst staining of SiHa cells after treatment with increasing concentrations of PQ showed increased cellular reactive oxygen species (ROS) levels, (B) MitoSOX and Hoechs staining show increased mitochondrial ROS in cervical cancer cells, and (C) increased
Intracellular ATP determination. Cells were seeded in 6-well plates at 2×10 5 cells/well and treated with PQ for 24 h. Adenosine triphosphate (ATP) were determined using the ATP determination Kit (Thermo Fisher Scientific, Waltham, MA, USA). Luminance   cervical cancer and QSG-7701 normal liver cells (used as an experimental control) were treated with 5-FU (60 μg/ml) and PQ in various concentrations (0, 20, 30, 40, 50 and 60 μg/ml) for 24 h before the MTT assay. The results showed that both 5-FU and PQ have strong cytotoxic effects on SiHa cervical cancer cells with higher levels exhibited by PQ compared to the 5-FU treatment, while both of them have a little cytotoxic effect on QSG-7701 normal liver cells ( Figure 1A). To determine whether PQ can induce cell apoptosis, the SiHa cells were treated with PQ (0, 40 and 60 μg/ml) for indicated duration times (0, 6, 12 and 24 h) and were stained using the Annexin-V FITC and PI detection Kit. The apoptotic cells using recombinant Annexin V conjugated to green-fluorescent FITC dye, PI stains necrotic cells with red fluorescence. After treatment with both probes, apoptotic cells show green fluorescence, dead cells show both red and green fluorescence, and live cells show little or no fluorescence. The results revealed that PQ treatment can significantly increase the cellular apoptosis in a time-and dose-dependent manner in SiHa cervical cancer cells ( Figures 1B and C). Furthermore, as shown in Figure 1D, PQ treatment significantly inhibited the cell migration and colony formation in SiHa cervical cancer cells.

PQ treatment accelerates the cellular and mitochondria ROS levels and mitochondrial dysfunction.
In order to detect whether PQ can increase intracellular ROS, SiHa cervical cancer cells were treated with PQ in various concentrations (0, 40, 60 μg/ml) for 24 h and the cellular ROS levels were detected by DHE & Hoechst staining. The results showed that PQ can significantly increase the intracellular ROS contents in SiHa cells (Figure 2A). At the same time, MitoSOX (a mitochondiral ROS indicator, which can be oxidized by superoxide in live cells) & Hoechst staining showed that PQ can also effectively increase ROS levels specifically in mitochondria ( Figure 2B). Higher mitochondria ROS levels will cause the disruption of mitochondrial function. In order to determine whether PQinduced ROS leads to oxidative stress that can cause a shift in the redox balance of mitochondria, we examined the increase of mitochondrial permeability in SiHa cells using the permeability of JC1 as a readout. JC-1 is a membrane permeable dye that selectively enters the mitochondria and can detect the intracellular mitochondrial membrane potential. When the mitochondrial membrane potential is high, JC-1 accumulates inside the mitochondria forming aggregates, which can generate red fluorescence; when the mitochondrial membrane potential is low, JC-1 cannot be gathered in the matrix of the mitochondria, it remains a monomer and produces green fluorescence (28). The results showed that the ratio of green fluorescence (low ∆Ψm) to red fluorescence (high ∆Ψm) of JC-1 in SiHa cells was significantly increased in various concentrations (0, 40, 60 μg/ml) of PQ treatment ( Figure 2C).
In addition to mitochondrial membrane potential, ATPsynthesis ability is also a fundamental indicator evaluating mitochondrial function (29). Therefore, we examined the intracellular ATP contents of SiHa cells treated with different concentrations (0, 40, 60 μg/ml) of PQ for 24 h. The results showed that PQ treatment significantly inhibited the ATP synthesis in SiHa cells ( Figure 2D).
PQ induces mitochondria-dependent apoptosis and MAPK signaling pathways in SiHa cells. In order to detect whether PQ is responsible for mitochondrial-dependent apoptosis, we treated SiHa cells with PQ (60 μg/ml) for the indicated duration times (0, 1, 3, 6, 12 and 24 h), and we then measured the protein levels of different caspases and their upstream B-cell lymphoma-2 (Bcl-2) family proteins. The results showed that the expression levels of cleaved-caspase3, cleaved-caspase9 and Bax proteins were upregulated in PQ-treated SiHa cells, while the Bcl-x L proteins were down-regulated following PQ treatment ( Figure 3A).
MAPK family members, including c-Jun N-terminal kinase (JNK), ERK1/2, and P38 MAPK, are involved in apoptosis (30). Following the same treatment as above, we assessed protein expression level changes of the MAPK signaling pathway. Our results showed that PQ treatment significantly increased the p-P38, p-ERK and p-JNK protein expression levels in SiHa cells ( Figure 3B). To understand the role of MAPK in PQ-induced cell apoptosis, the SiHa cells were tested using Annexin-V-FITC/PI staining after they were pretreated with inhibitors of MAPK, such as i) SB203580 (P38 inhibitor, 50 nM), ii) PD98059 (ERK inhibitor, 10 μM) and iii) SP600125 (JNK inhibitor, 40 nM) for 30 min followed by treatment with PQ for 24 h. The results showed that suppressing P38 with SB203580 significantly decreased the PQ-induced cellular apoptosis in SiHa cells but not ERK or JNK inhibitors (Figures 3C and D).

Inhibition of intracellular ROS reduces mitochondrial damage, cellular apoptosis, cell migration and colony formation in SiHa cells.
To detect whether ROS is a key molecule mediating PQ-induced apoptosis, the SiHa cells were pre-treated with N-Acetylcysteine (NAC, ROS inhibitor, 5 mM) for 30 min followed by treatment with PQ (60 μg/ml) for 24 h. The cellular and mitochondrial ROS levels as well as the mitochondrial membrane potential were detected using DHE, MitoSOX and JC-1 staining, respectively, and fluorescence microscopy. The results showed that treatment with NAC significantly reduced the PQ-stimulated cellular and mitochondrial ROS levels as well as the mitochondrial membrane potential in SiHa cells (Figures 4A-C). Furthermore, as shown in Figure 4D, NAC treatment significantly restored the PQ-induced inhibition of ATP synthesis. We also examined the effect of NAC on PQ with regard to the cell viability, apoptosis, migration, proliferation, in vivo 34: 1823-1833 (2020) as well as apoptosis-related protein expression using CCK-8 assay, Annexin-V staining, wound healing assay, colony forming assay and western blot in SiHa cervical cells, respectively. The results showed that scavenging of ROS with NAC treatment significantly restored the PQ-induced increase in cytotoxicity and apoptosis ( Figures 5A-C) in SiHa cells, and also recovered the inhibition of cell migration and proliferation ( Figure 5D). Western blot analysis showed that NAC could dramatically restore the PQ-induced up-regulation of pro-apoptotic c-cas3 and c-cas9 and down-regulation of anti-apoptotic Bcl-xL proteins expression ( Figure 5E).

Discussion
Cancer is a complex process involving a series of proliferative signals, including escape from growth inhibition, resistance to cell death, and uncontrolled proliferation as well as increased invasion and metastasis of cancer cells. A great focus has been on the development of new anti-tumor drugs, including natural plant drugs and their derivatives (6,31). In fact, herbal medicinal have been tested as potential treatment modalities for a variety of cancers, including leukemia, ovarian, testicular, lung, liver, esophageal, stomach, colon, and rectum cancer (32)(33)(34). In the present study, we reported that the P. quassioides extract bears anti-cancer properties in SiHa cervical cancer cells with a low cytotoxicity in normal cells, promoting apoptosis and inhibiting cancer cell migration and proliferation.
ROS, known as second messenger, play pivotal roles in cell survival, proliferation, migration and apoptosis (35). It was well known that mitochondria are not only the main source of intracellular ROS production but are considered as the main targets of ROS damage (36,37). In pathological conditions, excessive ROS formation in mitochondria can reduce energy synthesis, such as in the form of ATP, and accelerate the production of additional ROS (25,38), accumulation of which can damage the mitochondrial membrane and potentially lead to mitochondria-dependent apoptosis in cells (20,39,40).
In this study, our experimental data revealed that PQ treatment significantly increased intracellular and mitochondrial ROS production, as well as ATP synthesis accompanied by a decrease in the mitochondrial membrane potential in SiHa cervical cancer cells. Members of the caspase and Bcl-2 families are involved in promoting mitochondria-dependent cell apoptosis (41,42). In our research, we found that PQ treatment significantly upregulated the pro-apoptotic proteins, such as cleaved-caspase9, cleaved-caspase3 and Bax expression levels in SiHa cervical cancer cells while down-regulated the anti-apoptotic protein, Bcl-x L . Furthermore, ROS inhibition significantly attenuated apoptosis as well as their migration, proliferation and mitochondrial permeability in PQ-treated SiHa cervical cancer cells. All of these findings strongly suggest that ROS is a key point in PQ stimulation of normal function in SiHa cervical cancer cells. Despite, this understanding, the role of PQ on the mitochondrial membrane potential as well as the molecular mechanisms responsible for the increase of cellular ROS levels remains to be addressed in future studies.
Downstream of ROS, the MAPK signaling is involved in many cellular responses, such as senescence, proliferation, differentiation, and apoptosis (41,43). Members of the MAPK signaling involve the P38, ERK and JNK, whose pathways have been linked to apoptosis in various cancer cells as well as in cervical cancer (44,45). Recently, it was reported that a Brevilin A induces apoptosis in Breast Cancer Cells through the activation the JNK and P38/MAPK (46), while inhibition of the ERK-MAPK efficiently prevented apoptosis in hepatocellular carcinoma cells (47). In the present study, we also reported that PQ treatment significantly up-regulated the phosphorylation of P38, suggesting that MAPK signaling is involved in the PQstimulated cell apoptosis in SiHa cervical cancer cells.
There are some limitations in this study. First, the extract from P. quassioides is not a compound, thus we can't ensure the correct component that is responsible for activating apoptosis in cells, and second, we have no direct evidence to understand the molecular mechanisms linking ROS, P38/MAPK and mitochondrial dysfunction. Further studies will give answers to these questions.
In conclusion, our results revealed that P. quassioides extract exerts anti-cancer properties by increasing cytotoxicity and apoptosis, and inhibiting migration and proliferation in SiHa cervical cancer cells. The disruption of these cellular functions was caused by the accumulation of intracellular and mitochondrial ROS which in turn activated the P38/MAPK and the mitochondria/dependent apoptosis signaling pathway. Our findings provide insight for P. quassioides as a potential therapeutic strategy for the treatment of cervical cancer.

Funding
This research was supported by the project for postgraduate to innovate scientific research in Heilongjiang Bayi Agricultural University (No: YJSCX2019-Y64), the Natural Science Foundation of Heilongjiang Province of China (QC2015121), and the scientific research team support plan of Heilongjiang Bayi Agricultural University (TDJH201904), China.

Conflicts of Interest
The Authors declare that they have no competing interests.

Authors' Contributions
YXG, YL, YHJ, TK, YDC and HNS performed the construct of the model and wrote the manuscript. YHH, JL, GNS, DPX, CXR, LYY, DSL, JSK, YJJ, and JK performed the data analysis, JL and YHP provided P. quassioides extract reagents, TK, YDC and HNS were substantial contributors to study conception and design. All authors read and approved the final manuscript.