ZD0473R, and A2780cisR cancer cell lines, with the aim of overcoming cisplatin resistance and side effects. Cytotoxicity was assessed by MTT reduction assay. Combination index (CI) values were used as a measure of combined drug effect. MALDI TOF/TOF MS/MS and 2-DE gel electrophoresis were used to identify protein biomarkers in ovarian cancer and to evaluate combination effects. Synergism from combinations was dependent on concentration and sequence of administration. Generally, bolus was most synergistic. Moreover, 49 proteins differently expressed by 2 ≥ fold were: CYPA, EIF5A1, Op18, p18, LDHB, P4HB, HSP7C, GRP94, ERp57, mortalin, IMMT, CLIC1, NM23, PSA3,1433Z, and HSP90B were down-regulated, whereas hnRNPA1, hnRNPA2/B1, EF2, GOT1, EF1A1, VIME, BIP, ATP5H, APG2, VINC, KPYM, RAN, PSA7, TPI, PGK1, ACTG and VDAC1 were up-regulated, while TCPA, TCPH, TCPB, PRDX6, EF1G, ATPA, ENOA, PRDX1, MCM7, GBLP, PSAT, Hop, EFTU, PGAM1, SERA and CAH2 were not-expressed in A2780cisR cells. The proteins were found to play critical roles in cell cycle regulation, metabolism, and biosynthetic processes and drug resistance and detoxification. Results indicate that appropriately sequenced combinations of cisplatin with artemisinin (ART) and oleanolic acid (OA) may provide a means to reduce side effects and circumvent platinum resistance.In the present study, cisplatin, artemisinin, and oleanolic acid were evaluated alone, and in combination, on human ovarian A2780, A2780 Ovarian cancer is one of the major causes of death in gynaecologic cancers  marked wArtemisia annua L), (Oleanolic acid (OA) is a natural pentacyclic triterpenoid  9], pre, pre9], nnua L),  11]. It. ItArtemcisR (A2780 made resistant to cisplatin), and A2780ZD0473R (A2780 made resistant to platinum compound ZD0473) human ovarian cancer cell lines. Selected combinations were chosen to determine their effect on cellular platinum accumulation and Pt−DNA binding levels, changes in protein expression (using 2-DE gel electrophoresis). Finally, MALDI -TOF-TOF MS/MS was used to identify the proteins. Various studies relate to drug efficacy alone and in combination, and the mechanisms of action.The current study aimed to examine efficacy from binary combinations of artemisinin and oleanolic acid with cisplatin in A2780, A2780cisR, and A2780ZD0473R cell lines, was identified as IC50 values. The data revealed , OA, and ART that caused 50% growth inhibition in A2780, A2780ealed in  that the50, ED75, and ED90 standing for concentrations applying respectively to 50%, 75%, and 90% growth inhibition, were determined for all combinations. In essence, ED50, ED75, and ED90 are equivalent to IC50, IC75, and IC90. Dm is the median effect dose and m is the exponent defining shape of the dose response curve. All drugs in combination were added using the following three modes of administration:  to the selected cell lines where 0/0 h, 0/4 h, and 4/0 h respectively mean both drugs are added at the same time; cisplatin was added first, followed by phytochemical four hours later, and phytochemical was added first followed by cisplatin four hours later. In regards to the A2780 cell line, the data show that CS and ART  were calculated using the formula developed by Chou and Talalay to determine whether the combined drug action was antagonistic, synergistic, or additive. A CI value < 1 means a synergistic effect, equal to one means an additive effect, and > 1 means an antagonistic effect of the combined drugs  ED50, ED and ART , using 0 and ART , using 0 and ART  using 0/ and ART  using 0/bination , using 0bination , using 0cisR), indicating that the presence of ART and OA have served either to increase the uptake or decrease the efflux or both.The data  show thaciR cell line, platinum-DNA binding level from combination treatments was always higher than those from CS alone, suggesting the presence of such a correlation. Moreover, the observed increase in CS-binding levels in the A2780ciR cell line from combination with ART and OA are consistent with the increase in CS-accumulation levels due to the treatments.The data reveal that the platinum–DNA binding levels resulting from the combination of CS with ART or OA  in A2780Proteomics has analysed causes of drug resistance, in terms of the changes in protein expression, and has argued that ART and OA would be able to regulate the expression level of those proteins. Thus, proteins in the A2780 and A2780cisR cell lines were inspected by 2-D gel electrophoresis and mass spectrometry to examine their association with platinum drug resistance, wherein the protein expression levels in the A2780cisR cell line were compared to what was found in its counterpart cell line to determine the proteins that were differentially expressed. The objective of the proteomic study, besides the determination of new ovarian cancer biomarkers, was to evaluate the effect of: CS and ART (using 0/0 h), CS and OA (using 0/4 h), CS and ART (using 0/0 h), and CS and OA (using 0/0 h) on the expression of those dysregulated proteins (differently expressed proteins). cisR as compared to the values found in the untreated A2780 cell line, and (2) treated A2780cisR cell line  as compared to the values that were detected in both untreated cell lines. In both situations, at the beginning, the A2780 cell line was used as a reference. Later, the A2780cisR cell line was used as the reference. Proteins were considered to have undergone significant changes in expression if the fold factor was ≥ 2.0. The proteins that were detected by MALDI TOF/TOF MS and were matched against the Mascot database (http://www.matrixscience.com) and the UniProtKB/Swiss-Prot database (http://www.uniprot.org/) [cisR cell lines alternatively were used as a reference, are illustrated in tables (http://www.proteome.org.au/). Theoretical isoelectric point (pI), subcellular location, mass spectrum, and matched peptides were obtained from the Mascot database (http://www.matrixscience.com) and Swiss-Prot database (http://www.uniprot.org/) [http://www.proteome.org.au/). In this study, 49 proteins out of 101 scored above 56, according to the Mascot parameter that protein scores above 56 are considered significant (see Mascot database (http://www.matrixscience.com), Swiss-Prot database (http://www.uniprot.org/) [http://www.proteome.org.au/). Accordingly, those proteins were investigated for their involvement in the development of platinum resistance, besides evaluating the effect of the selected drug treatments on their expression.ot.org/) , when A2ot.org/)  through ot.org/) , and APAcisR and A2780ZD0473R cell lines than against parent A2780 cell line, ART showed greater sensitivity towards A2780 and A2780cisR than A2780ZD0473R cells while CS showed lower activities towards A2780cisR and A2780ZD0473R cell lines than the parent cell line. It has been reported that most of the natural products can modify several factors involved in the oncogenic transcription such as; Nrf-2, Hh /GLI, STAT3, FoxM1, PPAR y, Wnt/β-catenin, HGFR, HIF1 α, AP-1 and NF-κB [cisR and A2780ZD0473R cell lines may be attributed to its apoptotic effect, including the enhancement of p38 MAPK, ASK1 and ROS, and its inhibition effect of signalling pathways such as; S6K, PI3K, mTOR, Akt and NF-κB [The results show that OA showed greater activity against A2780nd NF-κB . In contnd NF-κB . That OAnd NF-κB . These fcisR, where the Pt levels in the resistant cell line were found to be significantly lower [The results show that the synergistic  effect fly lower , suggestly lower . Anotherly lower . Moreovely lower .cisR cells and 8.5 times higher than that from CS alone. Furthermore, in A2780cisR cells, Pt level resulted from the synergistic 4/0 h combination of CS and ART was nearly two times greater than that from synergistic 0/4 h combination and 4.5 times greater than that from CS alone. Together, these findings provide further support for the hypothesis that the ART growth inhibition action is a sequence and cell type dependent. The results are in agreement with the findings of Gong et al., wherein they reported that ART action was a cell type dependent [cisR cell line, Pt level from synergistic combination of CS with OA using 0/0 h sequence was nearly two times greater than that from treatment with 0/4 h combination and 3.3 times greater than that from CS alone. Moreover, treatment with synergistic combination 4/0 h, CS accumulation was 1.3 times greater than that resulted from 0/0 h combination and four times greater than that from CS alone. It appears that intracellular accumulation of CS in the presence of OA is also sequence-dependent. Thus, the observed increase in CS accumulation could be attributed to either the ability of OA to increase the influx of CS or to decrease CS efflux or both. The findings are supported by those of Ziberna et al., wherein they reported that intracellular concentration levels of chemotherapeutic drugs were enhanced because of OA’s ability to inhibit the efflux transporters [The results showed that Pt accumulations  from comependent . On the sporters . The incsporters . cisR cells than in A278 cells. A possible explanation is that the presence of ART and OA might have modulated some of the platinum resistance mechanisms, such as decreases in platinum-DNA adducts formation [Generally, platinum-DNA levels were greater in the A2780ormation , increasormation . Anotherormation ; therebyormation . For insormation , reductiormation , and enhormation .pIs, might be attributed to the post-translational modifications, which was suggested to occur in 2-D gel electrophoresis-based proteomics studies [cisR cell line were divided into nine major groups as discussed in the next section, using A2780 and A2780cisR cell lines alternatively as a reference, and are illustrated in A total of 133 spots were identified, out of which 101 proteins were significantly altered in expression. Among them, 54 spots were successfully identified by MS. In some cases, mass spectral analysis showed that the same protein was named for more than one spot such as spots 16, 18, and 76 were assigned to hnRNPA2/B1, spots 17 and 31 were assigned to ENOA, spots 52 and 63 were assigned to EF2, and spots 36 and 96 were assigned to VIME. Assignment of different spots to the same protein that exists in multiple isoforms with different  studies ,25, thusMortalin, also known as GRP75 [cisR cell line as compared to the sensitive A2780 cell line, consistent with the earlier studies [cisR cell line, suggesting that the applied treatments have improved chaperone action towards greater cell kill.as GRP75 , a membeas GRP75 , cancer as GRP75 . In this studies ,29,30. TAPG2 is another molecular chaperone and stress related protein, also known as HSP74 [cisR cell line as compared to the sensitive A2780 cell line. Up-regulation of APG2 was previously reported in hepatocellular carcinomas [as HSP74 , is ubiqas HSP74 . In thisrcinomas . The elercinomas . The finBIP, also known as GRP78 [cisR cell line as compared to A2780 cell line. Treatment with selected combinations, over-restored BIP expression, indicating that the treatments have prevented BIP’s roles in cell protection and the tumorigenicity of cancer cells, and thereby increasing the death of cells exposed to CS, suggesting that the selected combinations can serve as alternative therapeutic methods for the resistance in ovarian cancer associated with abnormal BIP expression. Thus, it seems that BIP may possibly be playing a major role in these synergistic outcomes.as GRP78 , was oveas GRP78 . It enhaas GRP78 , includias GRP78 . In lineHSP90B, also known as HS90B [cis-resistant cell line as compared to A2780 cell line, consistent with the findings of earlier studies [as HS90B , is one as HS90B ,36. HSP9as HS90B . It exhias HS90B . In the  studies ,26,27. TGRP94 also known as ENPL [cisR cell line as compared to A2780 cell line. The result is consistent with the findings of earlier studies [ as ENPL , is a me as ENPL . However studies ,28,30. CCYPA is one of CyPs isoforms  [cis-trans isomerization of the peptide bonds [cis-resistant cell line as compared to the sensitive A2780 cell line, consistent with the findings of the earlier studies [as PPIA) . CYPA cade bonds ,40. CYPAde bonds . In cont studies ,30. The  studies . Thus, tP4HB is one member of the PDI family, and is also known as PDIA1 [cisR cell line as compared to sensitive A2780 cell line. The result is in accordance with the previous findings of the earlier studies [as PDIA1 . It has as PDIA1 . P4HB isas PDIA1 . Silencias PDIA1 . Thus, ias PDIA1 . In the  studies ,28,30. TERp57 is another member of the PDI family, also known as Erp60, PDIA3, and GRP58 [cis-resistant cell line as compared to the sensitive counterpart cell line. Thus, the result together with literature data, indicate that dysregulation of the ERp57 expression enhances drug resistance and cancer progression while its level of expression is highly dependent on cell and cancer type. Hence, ERp57 may serve as a beneficial diagnostic marker and therapeutic target. Treatment of A2780cisR cell line with synergistic combination of (CS and OA using 0/0 h) over-restored ERp57 expression while (CS and ART using 0/0 h) partially restored its expression, indicating a causal relationship between ERp57 expression and platinum resistance in ovarian cancer.nd GRP58 , is a chnd GRP58 . ERp57 pnd GRP58 . It reacnd GRP58 . Althougnd GRP58 , liver, nd GRP58 . It was nd GRP58 . SimilarPGAM1, also known as PGAMA [cisR cells, possibly due to very low concentration. Given the important role of PGAM1 in the energy production associated with cell growth and cell proliferations, suggesting that PGAM1 may possibly be serving as a potential biomarker and therapeutic means towards Pt-resistance in ovarian cancer. Treatment with synergistic combination of (CS and ART using 4/0 h) over-restored PGAM1 expression, while treatment with synergistic combinations of (CS and ART using 0/0 h) partially restored PGAM1 expression. Whereas, after treatment with synergistic combinations of (CS and OA using 0/0 h) and (CS and OA using 0/4 h) PGAM1 expression was below threshold of detection. The results imply that level of PGAM1 expression was extremely sensitive towards the treatments that inhibited its ability to produce energy to nourish cancer cells thereby increased their vulnerability to the platinum drug. Hence, restoration of PGAM1 can be a contributing factor responsible for apoptosis enhancement so that the level of its expression can be a means for drug resistance in ovarian cancer. Therefore, chosen combinations can be helpful in sensitizing resistant cells towards platinum drugs and decreasing their side effects.as PGAMA , is a glas PGAMA . PGAM1 was PGAMA . In the LDHB, also known as LDH-H [cisR cell line as compared to sensitive counterpart cell line. The findings indicate that aberrant LDHB expression is cell, stage and cancer type dependent, hence, targeting LHDB expression level would be beneficial diagnostic and therapeutic strategies. LDHB expression was partially/over-restored after treatment with the chosen combinations, supporting the idea that synergistic outcomes, besides other reasons are associated with enhancement of CS cytotoxic effects by ART and OA through LHDB restoration which have modulated its role in energy metabolism, suggesting a strong correlation between LDHB expression and apoptosis improvement.as LDH-H , is invoas LDH-H . LDHB waas LDH-H  and nasoas LDH-H . Howeveras LDH-H , and in as LDH-H . SimilarATPA, also known as ATP5A1, is a mitochondrial enzyme involved in the synthesis of ATP from ADP [cisR cell line as compared to its sensitive counterpart, possibly due to low concentration. The findings suggest that the dysregulation of ATPA level contributes to carcinogenesis event and targeting this protein might be of therapeutic importance. Treatment with synergistic combinations of (CS and OA using 0/4 h) and (CS and ART using 4/0 h) over restored its expression. ATPA expression after treatment with synergistic combinations of (CS and ART using 0/0 h) and (CS and OA using 0/0 h) was not detectable, suggesting that the nominated combinations are extremely effective in overcoming drug resistance in ovarian cancer model.from ADP . Over-exfrom ADP , anaplasfrom ADP , acute lfrom ADP , multidrfrom ADP , and resfrom ADP . Howeverfrom ADP , and chrfrom ADP . In thisATP5H “generates ATP from ADP in the existence of a proton gradient throughout the membrane.” [cisR cell line as compared to the level found in sensitive A2780 cell line. The data point to the contribution of ATP5H in cancer formation and suggest that ATP5H may serve as a possible diagnostic marker and molecular target. ATP5H expression after the treatment with synergistic combinations of (CS and OA using 0/4 h), (CS and ART using 4/0 h), (CS and OA using 0/0 h), and (CS and ART using 0/0 h) was not detectable, suggesting that ATP5H expression is very sensitive to the combinations. Hence, these combinations would in helpful in opposing resistance in ovarian cancer.mbrane.” . ATP5H imbrane.”  and MCF7mbrane.” . ConsistTPI also known as TIM [cisR cell line with synergistic combination of (CS and ART using 4/0 h) partially restored its expression. Whereas, treatment with synergistic combination of (CS and OA using 0/0 h) fully-restored its expression, suggesting a strong correlation between TPI expression and improved cell kill.n as TIM , is invon as TIM . TPI expn as TIM , prostatn as TIM , epitheln as TIM , and hepn as TIM . This isGOT1, alternatively named as AATC [cisR cell line as reference, suggesting GOT1 involvement in the development of drug resistance thus, it can be promising therapeutic and diagnostic tool. Treatment with synergistic combinations of (CS and ART using 0/0 h) partially-restored GOT1 expression. After treatment with synergistic combinations of (CS and OA using 0/0 h), (CS and OA using 0/4 h) and (CS and ART using 4/0 h), GOT1 expression was not detectable, suggesting that GOT1 is highly sensitive towards the treatments. Thus, it may play a major role in the synergistic outcomes, implying a causal relationship between GOT1 expression and drug resistance.  as AATC , catalys as AATC . Yu et a as AATC . Similar as AATC . The samNM23, relatively named as NDKA [cisR cell line as compared to the level found in sensitive A2780 cell line using A2780 cell line as reference, suggesting that down regulation of NM23 plays a role in apoptosis inhibition, pointing out that NM23 expression can be a prognostic marker and therapeutic target. Treatment with synergistic combinations of (CS and ART using 0/0) partially-restored NM23 expression. NM23 expression was not detectable after the treatment with the synergistic combinations of (CS and OA using 0/0 h), (CS and OA using 0/4 h) and (CS and ART using 4/0 h), suggesting that its expression is strongly sensitive towards the treatments. Thus, the combinations may be beneficial in overcoming drug resistance involving abnormal NM23 expression. as NDKA , is cons as NDKA . It is i as NDKA , includi as NDKA . NM23 le as NDKA  and naso as NDKA . LikewisIMMT, also known as Mic60 [cis-resistant cell line as compared to its sensitive cell line, suggesting its usefulness as biomarker for platinum resistance in ovarian cancer. Treatment with synergistic combinations of (CS and ART using 0/0 h) and (CS and OA using 0/0 h) partially restored IMMT expression, suggesting that IMMT may be is playing a role in those synergistic outcomes, given that with the elevation of IMMT expression cell death was increased. Generally, this could be partly attributed to OA and ART actions which adjusted the molecular pathways including increasing Bax, decreasing Bcl-2, up-regulating of the caspases, and down-regulating NF-κB pathway thereby increased cell sensitivity towards platinum compounds leading to a greater growth inhibition. Therefore, the selected combinations can be a helpful in overcoming drug resistance involved abnormal IMMT expression.as Mic60 , promoteas Mic60 . It was as Mic60 . SimilarKPYM, also known as PKM2 and PKM [cisR cell line as reference. The results indicate that KPYM expression is cell and cancer type dependent and its expression is potential diagnostic and therapeutic targets. Treatment with synergistic combinations of (CS and ART using 0/0 h) and (CS and OA using 0/0 h) partially-restored KPYM expression. Whereas, after treatment with synergistic combinations of (CS and OA using 0/4 h) and (CS and ART using 4/0 h) KPYM expression was not detectable, implying that KPYM is very sensitive to the indicated combinations, suggesting a strong connection between enhanced apoptosis and KPYM expression.  and PKM , catalys and PKM . It is a and PKM , that pl and PKM , through and PKM ,79. KPYM and PKM  and in c and PKM , breast, and PKM ,81. LikePSAT, also known and SERC [cisR cell line as compared to its parental A2780 cell line; this could be attributed to the detection issue or low concentration. After treatment, synergistic combinations of CS and ART (using 4/0 h) and CS and OA (using 0/0 h), PSAT expression was over-restored, suggesting that PSAT may be is participating in the final outcomes where the apoptosis initiation may perhaps be related to improvement of PSAT expression. Thus, the present data may point out PSAT as potential diagnostic and therapeutic targets.and SERC , catalysand SERC , besidesand SERC . The eleand SERC  and cleaand SERC . HoweverVDAC1, also known as porin 31HM [cisR as a reference. The findings suggest that the VDAC1 is involved in tumour progression and that it may serve as useful biomarker and therapeutic target. After treatment with synergistic combinations of (CS and ART 4/0 h), (CS and OA using 0/4 h), (CS and ART 0/0 h) and (CS and OA using 0/0 h) VDAC1 expression was not detectable, suggesting that VDAC1 expression is significantly sensitive to the treatments. The result suggests that the rise in apoptosis could be attributed to a significant down-regulation of VDAC1 expression, suggesting a direct association between them, indicating the effectiveness of the selected treatments in restoring VDAC1 expression towards greater cell kill.rin 31HM , regulatrin 31HM . It interin 31HM . High lerin 31HM . LikewisENOA also known as Enolase 1 [cisR cell line as compared to its sensitive cell line A2780. Similarly, four previous findings from the host laboratory reported the same finding [nolase 1 , is a menolase 1 . It facinolase 1 ,88. ENOAnolase 1 . However finding ,28,29,30VIME is class 3 of intermediate filaments family [cisR cell line as compared to its sensitive cell line A2780. The results indicate that VIME is playing a major role in cancer progression and reveal that VIME expression can be a valuable marker for various cancers. Treatment with synergistic combinations of (CS and OA using 0/4 h) over-restored its expression in the A2780 cell line. Likewise, treatment with synergistic combinations of (CS and ART using 0/0 h) and (CS and OA using 0/0 h) over-restored the VIME expression. It can thus be suggested that the synergism and observed improve in apoptosis is directly dependent on VIME manifestation. Where the treatments were effective in preventing its cell protection role, given that VIME provides resistance against stress and maintains cellular integrity [s family ,89. It is family . VIME iss family . ConsistCAH2 is involved in extracellular acidification [cisR cell line as compared to its sensitive counterpart using A2780 as a reference, which may be due to low concentration or detection problems. Altogether, the findings suggest that the aberrant expression of CAH2 plays a role in cancer formation, metastasis, and resistance to chemotherapy; thus, it can serve as a promising diagnostic biomarker and therapeutic target. CAH2 expression was extremely sensitive to the treatment with synergistic combinations of (CS and ART using 4/0 h), (CS and OA using 0/4), (CS and ART using 0/0 h) and (CS and OA using 0/0), which might have modulated its acidification regulation activity in response to hypoxic environment leading to decrease cell proliferation that ultimately has increased cell death. Therefore, the results suggest that the restoration of CAH2 expression can be one of the causal factors for apoptosis induction. Thus, chosen combinations can be effective in combating drug resistance in ovarian cancer involving abnormal CAH2 expression.fication . CAH2 wafication , gastricfication . In contfication  and hepafication . In thisVINC is located in cell-adherence junctions and in focal adhesions [cisR cell line as compared to its sensitive counterpart using A2780cisR cell line a reference, the result suggests the VINC expression as potential therapeutic and diagnostic approaches. Treatment with synergistic combinations of (CS and ART using 0/0 h) and (CS and OA using 0/0 h) partially-restored its expression in A2780cisR cell line. Whereas, after treatment with synergistic combinations of (CS and ART using 4/0 h) and (CS and OA using 0/4 h) the expression of VINC was not detectable because of the effect of treatments, which by restoring its expression, may be altered protein turnover towards a greater protein degradation thereby enhanced growth inhibition. Hence, the result suggests that the significant reduction in VINC expression can be a causative factor accountable for improved growth inhibition.dhesions . It infldhesions . Vinculidhesions . In thisGBLP, also known as RACK1 [cisR cell line as compared to sensitive A2780 cell line. GBLP expression was over-restored due to the treatment with the selected combinations, suggesting that GBLP restoration is playing a contributory role in enhanced apoptosis resulting from chosen combinations which might have adjusted its behaviour thereby hindered the cell movement cell division towards a batter growth inhibition. It is likely that the significant improve in GBLP expression is directly associated with increased cells receptivity, suggesting GBLP as valuable diagnostic and therapeutic target.as RACK1 , is a cyas RACK1 ,93, whicas RACK1 . GBLP waas RACK1 . In the p18, also known as COF1, is a member of COF family [F family . It contF family . Over-exF family ,98, pancF family , In thisEIF5A1, also known as IF5A1 [cisR cell line with synergistic combinations of CS and ART (using 0/0 h) and CS and OA (using 0/0 h) EIF5A1 expression was partially-restored, suggesting that the treatments were able to interrupt its role in protein biosynthesis, thereby prevented translation leading to cell death. Therefore, the results suggest a causal relationship between EIF5A1 expression and platinum resistance in ovarian cancer. Hence, the selected combinations can serve as useful treatments to sensitize cancer cells towards platinum therapy. as IF5A1 , is one as IF5A1 , which ias IF5A1 . EIF5A1 as IF5A1 ; for thaas IF5A1  and knocas IF5A1 . The finEF2 is one member of the elongation group, involved in protein translocation in the elongation stage [cisR cell line, as compared to the A2780 cell line. The findings, together with the important role of EF2 in protein biosynthesis, clearly support the idea that elevation of EF2 expression is possibly be involved in ovarian cancer progression and drug resistance. Hence, the results represent EF2 as a new promising diagnosis biomarker and therapeutic target. Treatment, with synergistic combinations of CS and OA (using 0/4 h) and CS and ART (using 4/0 h) partially-restored EF2 expression. After the treatment with synergistic combination of CS and ART (using 0/0 h) EF2 expression was over-restored. This suggests that the observed rise in apoptosis degree is attributed to improvement of the CS cytotoxic effects by ART and OA actions towards opposing the drug resistance in ovarian cancer, indicating a causal relationship between EF2 expression and platinum resistance in ovarian cancer.on stage . EF2 wason stage , and thaon stage . The resEFTU is another member of the elongation proteins family [cisR cell line, as compared to its sensitive counterpart, using A2780, was used as a reference, and, consistent with this, Srisomsap et al. reported that EFTU was not expressed in the HepG2 cell line [s family . It regus family . EFTU was family . In thisell line  AltogethEF1G is another member of the elongation proteins. Over-expression of EF1G was observed in gastrointestinal tract malignancies [cisR cell line as compared to the level found in the sensitive A2780 cell line. Taken together, the balance of evidence suggests that the EF1G expression is possibly cancer- or/and cell type -dependent, suggesting that targeting EF1G might be of therapeutic importance. Treatment with synergistic combinations of CS and ART 9using 0/0 h) and CS and ART (4/0 h) partially-restored EF1G expression, whereas, synergistic combination of CS and OA (using 0/4 h) over-restored its expression, suggesting that the restoration of EF1G by the treatments might have inhibited transformation by preventing translation of proteins during protein biosynthesis that have a direct role on cell growth reduction. Hence, EF1G expression may be is directly participating in the synergistic outcomes resulting from the combination of CS with OA and ART, where the improved apoptosis could be directly linked to EF1G restoration. Thus, selected combinations are effective in improving cancer cell sensitivity towards platinum drugs involved EF1G irregular expression.gnancies , acute mgnancies , prostatgnancies . Howevergnancies . In thisEF1A1 is the other member of this group, involved in protein synthesis during elongation stage [cisR cell line as compared to its sensitive counterpart A2780. The findings suggest that EF1A1 plays an important role in apoptosis inhibition and development of drug resistance. After treatment with synergistic combinations of CS and OA (using 0/4 h) and CS and ART (using 0/0 h) EF1A1 expression was over-restored. While treatment with synergistic combination of (CS and OA using 0/0 h) partially-restored its expression, suggesting a straightforward connection between the elevated growth inhibition and decreased EF1A1 expression. This clearly supports that targeting EF1A1 can be a promising means to increase cancer cells sensitivity towards platinum drugs. Hence, selected treatments can serve as alternative approaches to overcome Pt-resistance through improving EF1A1 expression.on stage , which ion stage . EF1A1 ion stage . It is oon stage . ConsisthnRNPA1, belong to mRNA processing proteins, which plays fundamental functions in gene expression regulation at translational and transcriptional levels [hnRNPA1, also known as ROA1 [cisR cell line as compared to the level found in A2780 cells when A2780 was used as a reference, indicating that hnRNPA1 can be useful cancer biomarker and therapeutic target. Treatment of with synergistic combinations of CS and ART (using 0/0) and CS and OA (using 0/0) over-restored its expression. While after treatment with combinations of CS and ART (using 4/0) and CS and OA (using 0/4) hnRNPA1 expression was not detectable. Hence, the result suggests that the selected combinations have affected hnRNPA1 actions in gene expression and DNA repair, thereby increased cells vulnerability to CS cytotoxic action, leading to a better cell kill.l levels , also arl levels . hnRNPA1 as ROA1 , is up-r as ROA1 . SimilarhnRNPA2/B1, also known as ROA2 [cisR when used as a reference). After treatment with selected combinations, hnRNPA2/B1 expression was partially and over-restored, suggesting that the restoration of hnRNPA2/B1 expression possibly has altered its translation and transcription action, and, thereby, increased cell death. Hence, the result supports the possibility of a causal relationship between hnRNPA2/B1 expression and the resistance in ovarian caused by CS treatment. as ROA2 , is anot as ROA2 . Up-regu as ROA2 , and bre as ROA2 . LikewisPGK1 is participating in the glycolytic pathway and contributing in angiogenesis events [cisR cells as compared to the level found in sensitive A2780 cell line when A2780cisR was used as a reference. Thus, it can be said that PGK1 and its signalling genes can serve as future prognostic markers. Treatment with synergic combinations of CS and ART (using 0/0h), CS and ART (using 4/0h), and CS and OA (using 0/4 h) partially-restored PGK1 expression, suggesting the treatments might have influenced PGK1 angiogenic function, thereby preventing nutrients and oxygen, leading to a greater cell kill. However, its expression was further up-regulated due to the treatment with synergistic combination of CS and OA (using 0/0 h). From the above findings, it remains difficult to clearly establish clear association between PGK1 expression and enhanced cell kill and synergistic outcome. Hence, further studies would be necessary to gain better insight.s events . PGK1 was events ,118,119.PRDX1, also known as PAG [cisR cells as compared to the level found in A2780 cells when A2780 was used as a reference. However, Ren, Ye et al. reported a “relatively weak expression” of PRX1 in laryngeal cancer (Hep2), liver cancer cell (SUN449) lymphoma leukaemia (KOPN63) and acute lymphoblastic leukaemia (MOLT-4) cell lines [n as PAG , is an an as PAG . Its funn as PAG . Up-regun as PAG . In thisll lines . The disPRDX6, also known as 1-Cys PRX, is a bifunctional enzyme [cisR cell line as compared to the level found in A2780 cells when A2780 was used as a reference, however, RDX6 was very sensitive to selected treatments, suggesting a direct association between promotion of apoptosis and PRDX6 level of expression. Given its bifunctionality, it is possible that targeting PRDX6 might be of therapeutic importance. l enzyme  where itl enzyme . It is il enzyme , playingl enzyme . PRDX6 wl enzyme , lung [1l enzyme , and oesl enzyme . In the Op18, also known as STMN1 [cisR cells as compared to its sensitive counterpart using A2780 as a reference. However, the result is in line with previous findings from the host laboratory using the same cell lines [as STMN1 , is a hias STMN1 . It playas STMN1 . Fang etas STMN1 . In contll lines ,28, suggMCM7, also known as CDC47 [cisR cell line as compared to the level found in sensitive A2780 cell line. Treatment with the synergistic combinations of CS and OA (using 0/0 h) over-expressed MCM7 expression. While treatment with the combination of CS and ART (using 0/0 h) fully-restored its expression. This could be partially attributed to ART inhibitory ability of the important cell cycle regulators, including CDK4, CDK2 [as CDC47 , is a meas CDC47  involvedas CDC47 . MCM7 waas CDC47 . In thisK4, CDK2 , cyclin K4, CDK2 , given tCLIC1, also known as NCC27 [cisR cell line was used as a reference. Treatment with synergistic combination of CS and ART (using 0/0 h) over-restored its expression, whereas, treatment with synergistic combination of CS and OA (using 0/0 h) further down-regulated CLIC1 expression. These obviously confusing findings suggest that CLIC1 may possibly not be playing any important role in those outcomes. However, taken into consideration CLIC1′s roles in chloride channel [as NCC27  is one oas NCC27 . In the  channel , cell ad channel , suggestRAN is a member of the Ras super-family [cisR cell line as reference. Silencing of RAN inhibited cell growth, or/and induced apoptosis in colon, nasopharyngeal, renal breast, and ovarian tumours [r-family , also knr-family . It promr-family . The eler-family . Similar tumours . The fin1433Z is another signal transduction protein, it is one isoform of the 14-3-3 family, which exhibits a pro-oncogenic role in multiple tumour types [cisR cells as compared to its sensitive counterpart, when A2780 was used as reference. Likewise, previous findings reported down-regulation of 1433Z in  paclitaxel-resistant as compared to its sensitive counterpart [Moreover, ur types . The 143ur types , includiur types . The 143nterpart , and in nterpart . Howevernterpart , head annterpart , hepatocnterpart . Overallnterpart , CDK4, Cnterpart , S6K, PInterpart  and enhanterpart . This hacisR cell line was used as a reference. Up-regulation of PSA7 expression was reported previously in colorectal cancer [Two subunits of proteasome PSA3and PSA7 were also found to be differentially expressed, are involved in the regulation of several crucial cellular process, including apoptosis, immune responses, DNA repair, protein quality control and cell cycle progression . PSA3 , KI , A2780, A2780cisR and A2780ZD0473R ovarian cancer cell lines were kindly provided by Dr Philip Beale , Sydney, Australia), Varian Cary 1E UV/VIS spectrophotometer, Varian SpectrAA240 atomic absorption (AAS) with GTA-120 graphite furnace tube atomizer at the host laboratory , 28% ammonia solution , AgNO3 , FCS, 5× RPMI1640 media, L-glutamine, and NaHCO3 (5.6%) , 25 cm2 cell culture flasks , carbon dioxide incubator , JETQUICK DNA Spin Kit , Tris (hydroxymethyl) aminomethane hydrochloride , CalcuSyn software version 2 , urea and CHAPS , thiourea and DTT . Melanie version 7.0 . glycerol and SDS , DeStreak reagent , criterion TGX Precast gel, iodoacetamide, carrier ampholytes, PROTEAN i12 IEF system, mineral oil, and 10 X tris/glycine buffer , agarose, wicks, ChemiDoc XRS imaging system, Bio-Safe Coomassie Stain, Ready-Strip™ IPG Strip, criterion Dodeca cell, protein assay standard II kit, bromophenol blue, Microplate Reader Model 3550, and automated cell counter  96 well flat bottom plates (Edward Keller), and KCl .CS, OA, ART, trypsin, HEPES, triton-X 100, PBS, MTT, DMSO, and NaOH , K2, incubated in 95% air, 5% CO2 at 37 °C. Cells were preserved in (log) growth phase in RPMI 1640 containing heat-inactivated fetal calf serum (10%), bicarbonate (0.112%), HEPES (20 mM), and glutamine (2 mM). A2780 cell line was subjected to increasing intensity of CS until it gained resistant (A2780cisR). Moreover, A2780 cell line was treated continuously with increasing strengths of ZD0473 to develop resistant to the drug [Cell culturing was carried out following the same procedure as described previously . Brieflythe drug .Cisplatin was dissolved in dimethyl formamide (DMF) followed by addition of Milli-Q water (at the ratio of 1:5) to give 1 mM stock solution. Oleanolic acid (OA) was dissolved in ethanol to attain 1 mM concentration. Artemisinin (ART) was dissolved in ethanol and Milli-Q (mQ) water (at a ratio 50:50) to attain 1 mM concentration.50 value) was obtained from the standard curve, representing the percentage of the cell viability versus the concentration.Stock solutions were subjected to serial dilutions to give final concentrations ranging from 0.16 to 200 μM, using 10% RPMI1640 medium. Cells at a density of 4000 to 6000 cells/well were treated with CS, OA, and ART, individually at four different concentrations, repeated three times in the same plate, and incubated for 72 h. Two controls were used; one contained cell and medium and the other contained . Cytotoxic effects of CS, OA, and ART alone at different concentrations were evaluated using MTT assay following the procedure described by Mosmann . Briefly50 values. The concentration ranges were: cisplatin: 0.13–2.11 μM, 1.29–20.61 μM and 1.67–26.78 μM; oleanolic acid: 6.80–108.80μM, 5.23–83.68 μM, and 2.17–34.72 μM; artemisinin 3.36–53.70 mM, 5.36–85.76 mM, and 7.27–116.35 mM for A2780 (parent), A2780cisR (cisplatin resistant) and A2780ZD0473R (ZD0473-resistant) cell lines respectively. Briefly, 100 μL of cells seeded in each well of 96-well plate were treated with CS, OA, and ART alone and in combination with CS using the chosen concentrations, following three sequential modes; , (Pt/Phyt: 0/4 h) and (Pt/Phyt: 4/0 h), non-treated cells served as control. The plates were incubated for 72 h with each experiment repeated three times. The inhibition of cell growth was determined using the (MTT) reduction assay as previously mentioned. The combination index (CI) value of two compounds in combination was determined according to the method developed by Chou and Talalay [Cells were treated with solutions of compounds alone and in combination at three different concentrations, generally at constant ratios of their IC Talalay . CI of < Talalay ,138. cisR cells with density of (5 × 106 mL−1) in each petri dish treated with CS alone and its binary combinations with OA and ART using fixed concentrations of their IC50 values, were incubated for forty-eight hours. Cells were collected and centrifuged at 3500 rpm for two minutes at 4 °C. Next, cell pellets were washed twice with 4 mL of cold PBS and centrifuged at 3500 rpm at 4 °C for two minutes. Cell pellets were then re-suspended with 0.5 mL of cold PBS. Then (1.5 μL) was taken from each sample and mixed with the same quantity of trypan blue to determine the cell survival fraction using the automated cell counter. The samples were then re-spun for two minutes at 10,000 rpm at 4 °C then cell pellets were stored at −20 °C until assayed.A2780 and A27806 cells.Cell pallets were lysed with 0.5 mL of 1% triton-X100 added to each sample and sonicated in ice for thirty minutes then centrifuged at 14,000 rpm for three minutes. Then 400 μL of the supernatant from each sample was used to determine cellular platinum levels by measuring the absorbance values using graphite furnace atomic absorption spectrometry. The platinum level in each sample was calculated as nmol of platinum per 5 × 10Cell pellet from each sample was re-suspended in 200 μL of PBS for DNA extraction using JETQUICK DNA Spin Kit following the protocol of the provider’s instructions  to evaluate the effect of the selected combination treatments on those proteins. The studies were based on 2-D gel electrophoresis and the use of Melanie 7.0 software. The ultimate objective was to identify protein biomarkers that might be associated with the resistance towards CS, using MALDI TOF/TOF MS/MS. The identified spots were matched against Mascot (http://www.matrixscience.com), Swiss-Prot database (http://www.uniprot.org/), at APAF (http://www.proteome.org.au/). “This work was undertaken at APAF the infrastructure provided by the Australian Government through the National Collaborative Research Infrastructure Strategy (NCRIS).”The proteomic studies were initially intended: (1) to determine the proteins that underwent changes in expression in A2780cisR (5 × 104) cells were seeded and incubated for 24 h. The next day, A2780cisR cells were treated with chosen drug combinations, namely CS and ART (using 4/0 h), CS and OA (using 0/4 h), CS and ART (using 0/0 h), and CS and OA (using 0/0 h); then all samples (including untreated samples from both cell lines) were incubated for 24 h. Cells then were collected, centrifuged , washed (PBS/5 mL), re-suspend (by PBS/1 mL), counted, and centrifuged . Following this, the pellets were lysed in 500 μL of: (501 µL) of 65 mM DTT, (24.04 g) of 8 M Urea, (2.00 g) of 4%CHAPS, (7.61 g) of 2 M thiourea, (5 tablets) protease inhibitor and (up to 50 mL) of mQ water. Subsequently, the pellets were lysed and centrifuged at  for thirty minutes. Next, the supernatant was collected and stored at −80 °C  through their distinct bulletin numbers . Briefly, the IPG strip (11 cm L× 3.3 mm W × 0.5 mm thick/3–10 NL pH gradient) was passively rehydrated in 180 μL of rehydration solution containing: 4% CHAPS 100 μL, 0.0002% bromophenol blue, 60 mM DTT, 8 M Urea, (15 mg/1 mL) DeStreak, 2 M thiourea, and 0.2% ampholytes with 200 µg of protein from each sample then the samples were kept overnight. Then IEF procedure was performed using PROTEAN i12 IEF system following the manufacturer’s running protocol ** [v/v, 2% DTT w/v, 50 mM Tris–HCl pH 8.8, 2% SDS w/v, and 6 M urea) then were kept on a rocker for fifteen minutes ** [v/v, 50 mM Tris–HCl pH 8.8, 2% SDS w/v, 2% IAA w/v, and M urea) solution and left on a rocker for fifteen minutes ** [w/v) SDS, pH 8.3, and 25 mM Tris). The Criterion Dodeca Cell was filled with 6 L of (6 X) Tris/Glycine/SDS . Before running the program, gel cassettes were sealed with a melted agarose ** [cisR cell line, treated A2780cisR cell with the following combinations: CS and ART (using 4/0 h), CS and OA (using 0/4 h) and CS and ART (using 0/0 h) and CS and OA (using 0/0 h), and were imported into Melanie 7.0 software and clustered into six groups for spots detection and quantification, following the product’s manual instructions  treated A2780cisR with (CS and OA), and (4) treated A2780cisR with (CS and ART), subsequently, spots were automatically analysed and compared across all the samples using A2780 cell line as a reference after that untreated A2780cisR cell line as reference then spots were quantified automatically. In this study, a two-fold or greater change in protein expression was considered significant. Based on this parameter, 101 spots out of 133 spots were selected and sent to APAF (Macquarie University) for protein identification using MALDI-TOF and Mass Spectroscopy through APAF (http://www.proteome.org.au/). The detailed methods of MALDI and MS were previously described [http://www.proteome.org.au/), using 4800 plus MALDI TOF/TOF Analyser (AB Sciex), spots were excised (spot cutter/ Bio-Rad), de-stained (ammonium bicarbonate / acetonitrile) and digested . Next, the peptides were extracted (0.1% TFA/C18 zip-tip) and yield (MALDI-MS). The most intense (8) peptide peaks (ions) from each spot were further fragmented by (CID system). Masses and intensities were measured by MS/MS (TOF-TOF) and matched against Mascot database . Monoisotopic peak lists were matched against Homo sapiens using Swiss-Prot database (http://www.uniprot.org/) [http://www.matrixscience.com/) and as previously reported .For protein separation, first-dimension IEF was performed following the technique of the provider’s manual * . These Btocol ** . The 2ndtocol ** . Brieflynutes **  Again, tnutes ** . Next, Iarose ** . Sequentarose ** . The gelarose ** . The pains  proteins were 2 ≥ fold differently expressed and were successfully identified by MS. Those proteins were shown to play a critical role in cell cycle regulation, pro-survival, and anti-survival pathways, suggesting their participation in platinum drug resistance. Most of the identified proteins are associated with molecular chaperones and stress, metabolism, and invasion and metastasis. It can, thus, be suggested that those identified proteins can be additional novel predictive factors for early detection to individualised therapeutic interventions, considering the differences of each histologic type to improve the overall survival rate along with preserving the quality of the patient life. Finally, this study suggests that these synergistic combinations were able to restore back most of the protein expressions in the A2780


Lannea barteri Oliv. (Anacardiaceae) is believed to have activity against gastrointestinal, neurological and endocrine diseases. Previous studies on this plant have revealed antimicrobial, anticholinestrase, anticonvulsant, antioxidant and anti-inflammatory activities. However, the anticancer potential of L. barteri has not been studied to date. The aim of this study was to evaluate the anticancer potential of hot and cold extracts and silica gel column chromatographic fractions of L. barteri leaf and stem bark. The extracts and fractions were tested for anticancer activity by using the crystal violet cell proliferation assay on four adherent human carcinoma cell lines—5637 (bladder), KYSE 70 (oesophagus), SiSo  and HepG2 (hepatic). The inhibitory concentration (IC50) of fractions IH, 1I, 2E and 2F were: 3.75 ± 1.33, 3.88 ± 2.15, 0.53 ± 0.41, and 0.42 ± 0.45 µg/mL against KYSE 70 and 1.04 ± 0.94, 2.69 ± 1.17, 2.38 ± 3.64, 2.17 ± 1.92 µg/mL against SiSo cell lines respectively. Fraction 2E showed weak apoptotic activity at double the IC50 and some sign of cell cycle arrest in the G2/M phase. Thus, phytoconstituents of L. barteri leaf and stem bark can inhibit the proliferation of cancer cell lines indicating the presence of possible anticancer agents in this plant.In western Africa ethnomedicine,  Lannea barteri is one of such plant that may be a source of anticancer agents.Medicinal plants used in folk medicine are a rich source of compounds with anticancer activities. Studies of traditional medicine have resulted in pharmacological entities that have good efficacy and different mechanisms of action from the existing orthodox medicines. It is believed that phytoconstituents are good alternatives to conventional drugs used in the treatment of diseases because they are inexpensive and may have less adverse effects . DespiteL. barteri is a dioecious tree with a spreading crown; it can grow from 5–18 meters tall , KYSE 70 (oesophagus), SiSo  and HepG2 (hepatic) as well as the toxic effect of the extracts on Artemia salina larva.Indeed n Guinea . L. bart Burundi ,5. The b Burundi . When coermifuge . The barermifuge . Phytochermifuge ,8. Previicrobial ,8, anticicrobial , anticonicrobial  activitiicrobial , wound hicrobial . OxidatiThe extraction processes yielded hot (h) and cold (c) dichloromethane (DCM) , methanol  and water  extracts. The yields of the various extracts are shown in Phytochemical analysis on the DLEc and DSbEc gave positive reactions for terpenoids and steroids, respectively.The chromatograms of the active fractions from the cold DCM extracts of leaf (1E–1J) and stem bark (2D–2I) revealed absorption peaks (Au) of different heights at varying wavelengths between (210-600 nm) and retention times. The chromatogram of fraction 1E revealed five prominent peaks between retention times 24 and 32 min . FractioFraction 2D gave three peaks with one sharp peak which eluted at 24.75 min . Fractiocorr. (%) values of DLEh, MLEh, MSbEh, WLEh and WSbEh for the various cancer cell lines are shown in DLEh and DSbEh inhibited the proliferation of all the cancer cell lines while the MLEh, MSbEh, WLEh and WSbEh showed no growth inhibition. The (T/C) corr. (%) values for the extracts that had activity are shown in DLEc and DSbEc extracts inhibited the proliferation of all the cancer cell lines tested while MLEc and MSbEc inhibited 5637 and SiSo cell lines only. The WLEc and WSbEc did not inhibit the proliferation of any of the cancer cell lines. The T/C The order of antiproliferation activity of DLEc and DSbEc on the cell lines was SiSo> KYSE 70 > 5637.corr. values less than 50% were thus selected for secondary screening.The extracts with T/C 50 values of the hot extracts  against the KYSE 70 and SiSo cell lines are shown in The ICp < 0.05) higher than that of DLEc. The MLEc significantly (p < 0.05) inhibited the SiSo cell line more than the MSbEc (The growth inhibitory effect of the DSbEc on the KYSE 70 and SiSo were significantly (he MSbEc .p < 0.05) difference between DLEh, DLEc and DSbEc with DSbEc showing the highest inhibitory activity  higher inhibitory activity on SiSo cancer cell line when compared to MSbEc  inhibited the proliferation of HepG2 cancer cell line. When compared, DLEh and DLEc did not produce as much inhibition of HepG2 cancer cell line proliferation as DSbEh and DSbEc. Their IC50 values of the inhibition of the various cancer cells proliferations are shown in shown in On the HepG2 cancer cell line, both the hot and cold DCM extracts of the leaf and stem bark inhibited the proliferation of this cell line D. DSbEh 50 values (p < 0.05) higher with DSbEh and DSbEc compared to DLEh and DLEc  were subject to silica gel column chromatography and the collected fractions again screened for anticancer activity. The results showed that fractions 1E and 2F produced a higher inhibitory effect on the proliferation of 5637 of cell lines than the other fractions tested . Fractio50 values. The IC50 value of fractions 1H (3.75 µg/mL) and 1I (3.88 µg/mL) were significantly (p < 0.001) lower than that of fraction 1K (20.21 µg/mL) when inhibition of cell proliferation produced by all the fractions against KYSE 70 cell line were compared  and 1J (8.22 µg/mL) were significantly lower than that of fraction 1K while the IC50 values of fractions 1E (10.37 µg/mL) and IF (11.89 µg/mL) were lower than that of fraction K but not significantly , 0.53 (2E), 0.42 (2F), 6.05 (2G), 7.76 (2H) and 15.58 (2I). There was significant (p < 0.0001) difference between the IC50 of fractions 2E and 2F compared to fraction 2I on their inhibition against KYSE 70 (p < 0.01) difference in the IC50 values of fractions 2D and 2G in comparison to that of fraction 2I while the IC50 of fraction 2F significantly (p < 0.05) differed from that of fraction 2H just as the IC50 of fraction 2H significantly (p < 0.05) differed from that of fraction 2I (The IC KYSE 70 B. There ction 2I B. p < 0.05) inhibition of SiSo cell line proliferation were recorded with fractions 1F, 1H, 1I, 1J and (p < 0.01) with fraction 1H compared to fraction 1K  when compared to fractions 1E and IK respectively; they had the lowest IC50 values of 0.25 and 0.30 µg/mL respectively when all the treatment groups were compared  when compared with other fractions  against HepG2. The inhibitory effect of the 2E and 2F fractions against the proliferation of HepG2 cell line were significant (p < 0.01) compared to fraction 2I , 19.9%, 16.38% (1H), 18.52%, 11.58% (1I), 31.09%, 11.74% (2E), 30.87% and 15.30% (2F) , 31.16%, 14.19% (1H), 19.95%, and 17.25% (1I), 47.07%, 34.00% (2E), 27.85% and 25.83% (2F) respectively. From this result, fraction 2E gave the highest fractions (47.07% and 34.00%) of apoptotic cells at doubled the IC50 concentration after 24 h and 48 h incubation periods, respectively, compared with other fractions and control. Meanwhile, the leaf fractions especially fraction 1I had the highest number (0.68 and 1.22%) of necrotic cells than the stem bark fractions at doubled the IC50 concentration after those incubation periods.The percentage of apoptotic cells induced by the fractions and DMSO at IC30% (2F) . At conc50 concentration of fraction 2E at 48 h incubation period compared to control  in the G1/G0 phase with concomitant increase in the population of cells accumulated in S (24.78%) and G2/M (20.04%) phases especially at doubled the IC control . Fractionificant .in vivo, the brine shrimp assay was used. In this assay, the lethal concentration at 50% of the population (LC50) of the DLEc was found to be 5649.20 µg/mL while the extrapolated effective concentration was 564.92 µg/mL. However, the LC50 of DSbEc was 124.78 µg/mL while the EC50 was 12.48 µg/mL. At 1000 µg/mL, the percentage death was high (66.7%) with DSbEc and low (13.3%) with DLEc. The calculated therapeutic index (TI) for both extracts was 9.99  of the DLEc that killed the 50% of the population of the larvae was higher than the concentration of DSbEc that killed the same percentage of the population. The concentration of the DLEc effective at 50% of the population (EC50) was higher than that of DSbEc even though they had the same therapeutic index. The EC50 of a quantal dose response curve represents the concentration of a compound where 50% of the population exhibits a response after a specific exposure time. It also measures the potency of substances [50 and LC50 values was more potent as toxic agent than the leaf extract.Brine shrimp lethality assay is an essential tool used for preliminary assessment of toxicity of medicinal plant products by measuring their potentials to kill larvae of na leach . It is abstances . TherefoL. barteri revealed some level of apoptotic activity and cell cycle arrest. Apoptosis is a form of programmed cell death in multicellular organisms that involves biochemical cascade of events leading to morphological changes such as  and eventual death without affecting the normal cell [Assessment of the mechanism of cytotoxic activity of the most active fractions from the leaf and stem bark extracts of mal cell . Apoptosmal cell . Fractiomal cell ,22.The cell cycle is a physiological process that occur in distinct stages to assure controlled cell growth, development and division. Cell proliferation and differentiation are under tight genetic control in normal cells. For cancer to arise there is altered control in the genetic machinery that controls cell proliferation and eventual progression of stages of cell cycle which is the hallmark of cancer growth and sustenance . Cell cyLannea barteri leaf and stem bark possess potent anticancer activity against 5637 (urinary bladder), KYSE 70 , SiSo (cervix adeno-) and HepG2 (hepatocellular) human carcinoma cell lines. The stem bark extract showed greater activity than the leaf extract. It would appear that apoptosis and cell cycle arrest at G2/M phase are likely anticancer mechanisms of action. The anticancer activity may be attributed to unknown phytoconstituents inherent in the plant. Work is ongoing to purify the most active fraction, isolate and characterize the compound(s) that have the anticancer activity, to test them on other cancer cell lines and to explore other possible mechanism(s) of cytotoxic activity.In conclusion, our findings show that the cold DCM extracts and fractions from the ® Entris, BP 21005, Goettigen, Germany), ultrasonicator , magnetic stirrer , rotary evaporator  with heating bath , folded qualitative filter paper 301 size 185 mm with particle retention size of 12–15 µm, flasks , measuring cylinders, mechanical grinding machine, refrigerator of−20°C, lyophilizer, analytical TLC plates  and silica gel , VLM EVA 2® Evaporator , column fractions retriever, 96–well microtiter plates and25 mL and 75 mL flasks , 8- and 12- channel–pipette , centrifuge (Heraeus Biofuge primo R) and incubator , particle counter and size analyzer , sterile filters , universal shaker, , HPLC system , microtiter plate reader  and flow cytometer .Digital and electronic weighing balance , ribonuclease-A (Rnase) and NaHCO3 , 50% glutaraldehyde solution, trypsin—EDTA solution and fetal calf serum (FCS) , HPLC gradient acetonitrile (ACN) , Dulbecco’s buffer (pH 7.4): KCl—0.2 g/L, NaCl—8 g/L, MgSO4.7H2O—0.1 g/L, KH2PO4—0.2 g/mL, Na2HPO4.7H2O—1.55 g/mL, AnnexinV apoptosis kit , propidium iodide , phosphate buffered saline (PBS), Triton X-100 .All solvents were analytical grade and water was purified either by distillation or with an Elga PureLab Flex4 , penicillin/streptomycin solution , RPMI-1640 culture mediumKYSE 70, SiSo, 5673 and HepG2 human cancer cell lines were obtained from the German Collection of Microorganisms and Cell Culture .Artemia salina larvae were obtained from Department of Veterinary Pharmacology, Faculty of Veterinary Medicine, University of Nigeria, Nsukka.Lannea barteri were collected from Benue State, Nigeria in the months of March and April by Mr. Alfred Ozioko of International Centre for Drug Discovery and Development and the specimen voucher (Voucher No: 096) deposited at the Centre’s herbarium. The plant materials were dried under shade, and subsequently pulverized into powder for future use.The fresh leaves and stem bark of All extraction solvents were distilled to their pure states according to their specific boiling points: 65°C (methanol), 39.8°C (dichloromethane (DCM)), 59°C (ethyl acetate) and 69°C (n-hexane).About 2 g each of the powdered material were subjected to successive cold extraction by using the following solvents in increasing order of polarity; DCM, methanol and water. The powdered material was soaked in about 100 mL solvent in a conical flask and then sonicated for 10 min in an ice pack to break the cell wall of the plant biomass. Afterwards, the flask was placed on a magnetic stirrer for 180 min at 250 rpm. The content of the flask was centrifuged at 3500 rpm for 10 min and the supernatant filtered into a round bottom flask. The extraction process was repeated two more times after which the filtrates were pooled together and evaporated by using a vacuum rotary evaporator in a fume hood according to the boiling pressure of the solvent: 880 mbar (DCM), 337 mbar (methanol) and 72 mbar (water) at 50°C. After extraction with each solvent, the marc was dried overnight in a fume hood before the next solvent was used until the last solvent. The concentrated extracts yielded cold (DCM) leaf (DLEc) and stem bark (DSbEc) extracts, cold methanol leaf (MLEc) and stem bark (MSbEc) extracts and cold water leaf (WLEc) and stem bark (WSbEc) extracts which were weighed, yield in (%) calculated and stored in the refrigerator for future use. The extracts were subjected to anticancer screening by the crystal violet cell proliferation assay as a bioactivity guide (see below).About 10 g of the plant material was extracted successively with these solvents in the increasing order of polarity according to their boiling temperatures 39.8°C (DCM), 65°C (methanol), and 100°C (water) in continuous extraction for 12 h by using a Sohxlet extraction apparatus. After extraction with each solvent, the marc was dried overnight in a fume hood before the next solvent was used until the last solvent. The extracts were concentrated using rotary evaporator as described in The extracts were subjected to anticancer screening by the crystal violet cell proliferation assay as a bioactivity guide (see below).The DSbEc and DLEc which exhibited the greatest antiproliferative activity were subjected to TLC by using 1:1 ratio of analytical grade n-hexane and ethyl acetate and the retention factors were calculated .corr. (%) values were selected for secondary screening using crystal violet cell proliferation assay and determination of IC50. The fractions were then subjected to HPLC analysis.The DLEc and DSbEc were subjected to column fractionation on 60 H 70–230 mesh silica gel by using gradient elution method with these respective solvent combination ratios (9:1 (200 mL), 1:1 (200 mL), 2:8 (200 mL)) of analytical grade n-hexane and ethyl acetate; and lastly 100% methanol (100 mL). The resulted fractions (1A-K from DLEc and 2A-2J from DSbEc) were further subjected to primary screening with the crystal violet cell proliferation assay. The seven fractions (1E-K) from DLEc and six fractions from DSbEc (2D-I) that gave the lowest T/C The cytotoxic fractions (50 mg/mL) mentioned in The DLEc and DSbEc were screened for the presence of bioactive components following the methods of ,26.corr. (%) was calculated with the equation:T is the mean OD of the treated cells, ODC the mean OD of the controls, and ODC,0 the mean OD of seeded cells at the time the drug was added. The crystal violet cell proliferation assay was used as a bioactivity guide in both primary and secondary screening assays as previously described ,28. To p50  values were estimated by linear least – square regression of the (T/C) corr. (%) by using MS Excel® function trend values versus the logarithm of the concentration of extract. The concentration that showed (T/C) corr. values between 10% and 90% were used in the calculation. The reported IC50 values are the average of 3 independent experiments. The IC50 value indicated the potency of the extract and equaled the amount of complex that inhibited the cell growth by 50%. The lower the IC50 value the greater the potency of the extract.The ICcorr. values for the extracts were calculated as described above.Cells were treated with stock solution of the extracts dissolved in DMSO at 50 mg/mL; a 10 µL of the stock solution was added to 5.0 mL of RPMI medium containing 10% FCS and 1% penicillin/streptomycin solution, of which 100 µL of the solution was added to each well to give final concentrations of 50 µg/mL extract mass.. One plate was left untreated for each cell line and this served later as the control (C) at time zero (0)  when results were obtained. The plates at time zero were stopped as previously described above. Treated cells were returned to the incubator for 96 h at 37°C and stored after fixation with GBS in the refrigerator at 4°C until required . The T/C50 values of the extracts estimated.Two-fold serial dilutions of the 50 mg/mL stock solution were made by diluting with DMSO to obtain five stock dilutions. The stock solution was diluted 1:500 with the culture medium and 100 µl were added to cells in 96-well plate as triplicates starting with the least concentration to the highest concentration . The plaThe toxic effect of DLEc and DSbEc on brine shrimp larvae was measured by using a previously reported method ,30.Fractions 1H, 1I and 2E, 2F were selected for apoptotic and cell cycle analysis with the SiSo cell line.The induction of apoptosis of the fractions on SiSo cell line was determined by using an Annexin-V/propidium iodide double staining, flow cytometric method as described previously .The ability of the fractions to arrest the cell cycle progression in SiSo cell line was done by staining cells with propidium iodide and measuring the phases of the cell cycle by flow cytometry as described previously .A summary of the extraction, fractionation and testing procedures is shown in


One of the most important and striking characteristics of hepatocellular carcinoma (HCC) with intrahepatic metastasis, is that it results in extremely poor prognosis. Animal models have become a fundamental and very useful in research for disease study. However, some limitation has arisen from these model systems. We have therefore established a model of HCC with intrahepatic metastasis and noticed some differential appearances in different HCC cell lines. Luciferase-transfected HCC cell lines MHCC97-H and PLC/PRF/5 were inoculated into SCID mice via spleen. Observation the intrahepatic metastasis by bioluminescence imaging in vivo and comparing of the differential formation of metastatic lesions between different HCC cell lines by incorporating physical anatomy was done. Animal models for HCC intrahepatic metastasis were well established. However, there were some clearly noticed differences between MHCC97-H and PLC/PRF/5 cell lines. The group of MHCC97-H cell line readily metastasis in the liver, whereas group PLC/PRF/5 cell line developed extensive intrahepatic metastasis and formed large tumor in situ in the spleen. MHCC97-H and PLC/PRF/5 cell lines can be used to successfully establish a model of HCC intrahepatic metastasis with distinctive characteristics, which provides an important direction for the study of the mechanism of HCC intrahepatic metastasis, and may hopefully provide a basis for clinical treatment. Surgery has become the most effective therapeutic strategic method in managing HCC patients, but the recurrence rate is still as high as 80%, and most of this recurrence occurs within 1–2 years post operatively2. The most common form of HCC recurrence comes as a result of intrahepatic metastasis. The reresection rate after recurrence is up to 10.4–31%, but the 3-year survival rate after reresection is only 52.6%3. Therefore, recurrence and metastasis are the main factors affecting the survival rate and contributing to prognosis in HCC patients. The exact mechanism under HCC recurrence and intrahepatic metastasis need to be explored by the utilization of animal models, however, the appropriate animal model of intrahepatic metastasis is not well established and verified.Hepatocellular carcinoma is the most common disease with malignant tumors of the liver and the third resulting in cancer mortalities in the world4 or tumor tissues5 into the liver and then form metastasis, which is most similar to the in-vivo condition of HCC intrahepatic metastasis. However, the application of this animal model is limited due to the destruction of the original morphological structure of the liver and the difficulty in arresting and controlling bleeding. There is also a long waiting time for formation and progression of intrahepatic metastasis. Secondly, an injection administration of HCC cells via portal vein is also an ideal method for intrahepatic metastasis. Nevertheless, it is difficult to perform an operation and arrest bleeding and this gives rise to a lower postoperative survival rate6. Finally, the injection administration of HCC cell into spleen is widely used to establish intrahepatic metastasis model due to its simple operation and high rate of tumor formation. It was found that the hepatoma cell suspension being injected into the spleen not only formed the tumor in situ, but also the definite metastasis in the liver8.There are several HCC intrahepatic metastasis animal models. First and foremost, directly implanted cell lines9, produces HBsAg. PLC/PRF/5 is moderately invasive and be widely used in basic research. Studies have found that HBV and AFB1 (Aflatoxin B1) induces and gives rise to the development of HCC. Human HCC cell line MHCC97-H appears as polygonal epithelial cells with metastatic potential, which can secrete AFP. It has been found that these cell lines after intrahepatic inoculation can lead to liver, diaphragm and abdominal wall invasion11. In this study, we used PLC/PRF/5 and MHCC97-H HCC cell lines to establish HCC intrahepatic metastasis models and observed the differences between both cell lines. Our study paved a way for the fundamental research of HCC intrahepatic metastasis.HCC cell line PLC/PRF/5 which was established by Alexander et al.Severe combined immunodeficiency (SCID) CB-17 mice purchased from Weitonglihua  were used in our study. All animals were housed in a SPF condition in our lab. Mice were operated at 6–8 weeks and their body weight ranged from 18 to 22 g. All animal experimental procedures were in accordance with ARRIVE guidelines.HCC cell lines MHCC97-H and PLC/PRF/5 were cultured in DMEM  supplemented with 10% FBS (Invitrogen) and 1% penicillin (Invitrogen) in an incubator under a 5% carbon dioxide atmosphere at 37 °C in a relative humidity of 95%.5 per plate and prepared of transfection. Multiplicity of infection (MOI) of PLC/PRF and MHCC97-H are 10 and 30 respectively. Then viruses were transfected into both cells and using polybrene (10 ug/ml) to enhance transfection efficiency. After 12 h of incubation, the medium was replaced with a fresh DMEM complete medium. After incubation for 48–72 h, cells were observed by an in vivo imaging system by adding d-Luciferin, Sodium Salt to evaluate the efficiency of transfection.Luciferase negative control virus purchased from Genechem Chemtech  was utilized in our study, and the sequence was 5′-TTCTCCGAACGTGTCACGT-3′. Virus transfection were operated as follows. PLC/PRF/5 and MHCC97-H cells were cultured in 6-well plates at a concentration of 2 × 106/100 ul complete medium, which were completely injected into spleen within a minute. Postoperatively all mice were placed on a warm thermal generator until they were fully restored to a normal condition. Mice were finally kept under close observation and monitoring under SPF conditions.All animal studies complied by incorporating National Institutes of Health guidelines for the care and use of Laboratory animals were approved by the Lanzhou University Second Hospital Institutional Animal Care and Use committee (D2017-052). Mice were grouped into PLC/PRF/5 group (n = 3) and MHCC97-H group (n = 6) respectively. All cells transfected with virus were used and injected into spleen of mice using an insulin needle. The cellular concentration was 1 × 10d-Luciferin, Sodium Salt. After 10 min, mice were anesthetized with isoflurane gas and placed into the imaging chamber and X-ray imaging was performed. Post X-ray shooting bioluminescence imaging was done.In vivo Bioluminescence assays were performed on days 4 and 14 postoperatively. All mice were intraperitoneally injected with 150ul 15 mg/ml P value was < 0.05.For the statistical analysis SPSS22.0 software was used. Numerical data was analyzed using the Student’s t test. A difference was considered significant when the After transfecting with luciferase virus, the efficiency of transfection was evaluated by the utilization of bioluminescence and luciferase assays. Results showed that PLC/PRF/5 Fig. c and MHCIn vivo Bioluminescence assays were first performed on day 4 post-operation, which indicated that both groups PLC/PRF/5 Fig. a and MHCP < 0.05)  Fig. e and at 05) Fig. g.Most importantly, we observed that there was a vast difference of gross specimen of the spleen between the two groups. As it could be seen, huge in situ tumors were formed in the spleen of PLC/PRF/5 group Fig. h, but thIntrahepatic multiple metastasis and recurrence are typical features and signs of HCC, which always lead to very poor disease prognosis in these patients. However, the mechanism of this phenomenon is still very unclear. The high incidence and the unclarity of HCC intrahepatic metastatic disease process and manifestation has made researchers to come up with an appropriate animal model to try and understand the disease process.First of all, we established HCC intrahepatic metastasis model by injecting suspended HCC cell lines into spleen. This method is not only simple to operate, but has a high rate of tumor formation. However, it leads simply to, effective bleeding and difficulties in coagulation processes since the spleen is a very fragile organ. Furthermore, we transfected control virus with luciferase into HCC cell lines, which was convenient to evaluate the distribution and growth of tumors in vivo in real time.We selected two HCC cell lines with different degrees to explore their potential for intrahepatic metastasis respectively. In comparison, MHCC97-H is a highly metastasis potential cell line, while PLC/PRF/5 is not. After operation we performed an in vivo Bioluminescence assay on days 4 and 14 post-operation, which revealed that both of cell lines could form tumors in vivo and multiple intrahepatic metastasis lesions were also clearly observed. This meant that both cell lines had a strong tumor-forming potential and could be used to establish animal models of HCC.8. Therefore, we speculate that PLC/PRF/5 cells are more likely to adhere and have adhesion properties than MHCC97-H cells. However, it is not clear at the present moment what really contributes to PLC/PRF/5 cells to have a stronger ability of adhesion and further research is needed to elucidate this phenomenon.Next, we compared the difference in tumor formation between the two cell lines. First of all, it could be clearly noticed that MHCC97-H group had a longer survival time than PLC/PRF/5 group at the 25 and 35 day respectively. So, we dissected the mice at different time and found out that the livers of PLC/PRF/5 group were significantly heavier than those in the MHCC97-H group. Most importantly, huge tumors in situ were formed in the spleen of PLC/PRF/5 group, while there was almost no tumor formation in the spleen of MHCC97-H group. A study comparing the growth capacity of transplantable tumors in several organs found that two of the hepatomas grew much more readily and vividly in the spleen than in any other organs elsewhere. They suggested that the difference in their capacity to metastasize is related to the differences in cell adhesiveness. The spleen seems to be structurally well adapted to permit the release of non-adhesive cells into the blood streamIn summary, the SCID mice model established here have similarities with HCC intrahepatic metastasis patients. HCC cell lines in both MHCC97-H and PLC/PRF/5 are tumorigenic and have a great potential for intrahepatic metastasis. In addition, the two cell lines are different in their spleen appearance, while the specific mechanism is not explicit and needs further investigations and study. Overall, it is definite that our model system will be a useful tool for the basic research of intrahepatic metastasis of HCC and can well provide researchers with the broad base of cell lines selection.Supplementary file1


Breast cancer (BC) is the most common malignancy among women. Emerging studies have demonstrated that circular RNA (circRNA) zinc finger RNA binding protein (circZFR) serves as a crucial regulator in many human cancers. However, the role and mechanism of circZFR in BC tumorigenesis remain unclear.The levels of circZFR, miR-578 and hypoxia-inducible factor 1α (HIF1A) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) or western blot. Cell viability, colony formation, apoptosis, migration and invasion capacities in vitro were determined by using the Cell Counting Kit-8 (CCK-8), standard colony formation, flow cytometry and transwell assays, respectively. Glucose uptake, lactate product and adenosine triphosphate (ATP) levels of cells in vitro were measured using the commercial human assay kits. Targeted relationships among circZFR, miR-578 and HIF1A in BC cell lines were verified by dual-luciferase reporter and RNA pulldown assays. Animal studies were performed to assess the effect of circZFR on tumor growth in vivo.Our data indicated that circZFR was overexpressed in BC tissues and cells, and the increased circZFR level predicted poor prognosis of BC patients. CircZFR silencing or miR-578 overexpression repressed BC cell viability, colony formation, migration, invasion, and glycolysis and enhanced cell apoptosis in vitro. CircZFR silencing also hampered tumor growth in vivo. Mechanistically, circZFR acted as a sponge of miR-578, and circZFR silencing hindered BC cell malignant behaviors by miR-578. HIF1A was a functional target of miR-578 in regulating BC cell viability, colony formation, migration, invasion, glycolysis and apoptosis in vitro. Furthermore, circZFR modulated HIF1A expression through sponging miR-578.Our findings first identified that the silencing of circZFR suppressed BC malignant progression in vitro via the regulation of the miR-578/HIF1A axis, providing evidence for the crucial involvement of circZFR in BC pathogenesis. Breast cancer (BC) remains the most commonly diagnosed cancer and the leading cause of cancer-associated death among females in 2018 . TherefoCircular RNAs (circRNAs) are covalently closed, endogenous RNAs that have crucial non-coding functions in human physiologic and pathologic processes . Work inMiRNAs modulate gene expression but are frequently dysregulated in human cancers, including BC . Danza eHere, we undertook to examine the biological effect of circZFR in the malignant progression of BC in vitro and in vivo. We identified that circZFR, a prominently up-regulated circRNA in BC, controlled BC progression in vitro via targeting the miR-578/hypoxia-inducible factor 1α (HIF1A) axis.In this study, we enrolled 70 BC patients admitted to The First Affiliated Hospital of Zhengzhou University from April 2013 to June 2014. The clinicopathological features of these patients were provided in Table ®HTB-22), BT-549 (ATCC®HTB-122), MDA-MB-231 (ATCC®HTB-26) and MDA-MB-453  were cultured in RPMI-1640 medium, supplemented with 10% fetal calf serum (FCS) and 1% antibiotic solution . The immortalized MCF10A cell line  was maintained in Dulbecco’s modified Eagle’s medium/Nutrient Mixture F-12 (DMEM/F-12) with 10% FCS, 20 ng/mL epidermal growth factor, 0.5 μg/mL hydrocortisone and 5 μg/mL insulin . All cells were cultured in a 5% CO2 incubator at 37 °C.BC cell lines MCF7  synthesis was done using total RNA (100 ng) isolated by Isogen  from tissues and cell lines. The levels of circZFR and HIF1A were quantified using the TaqMan Gene Expression Assays with the indicated primers, and mature miRNAs were assayed using the TaqMan MicroRNA Assays with TaqMan-specific primer probes as recommended by the manufacturers . qRT-PCR was run in triplicate on the iCycler iQ5 device  using the PCR conditions as previously reported . Resultst method . Primer t method .CircZFR knockdown in MCF7 and BT-549 cell lines was achieved by the transduction of corresponding lentiviruses expressing three different sequence shRNAs specific to circZFR (sh-circZFR#1 (sh-circZFR), sh-circZFR#2 and sh-circZFR#3, Fulengen, Guangzhou, China), and nontarget shRNA lentiviruses (sh-NC) were used as the negative control. Vector-transduced cells were selected by puromycin  at a final concentration of 2.5 μg/mL at least 72 h. MiR-578 overexpression and knockdown cell lines were generated using the synthetic miR-578 mimic  and inhibitor , respectively, with a corresponding nontarget oligonucleotide  as the negative control. To elevate HIF1A expression in BC cell lines, a recombinant overexpressing plasmid for HIF1A  or negative control plasmid  was transiently transfected into cell lines using Lipofectamine 3000 reagent  as per the manufacturers’ protocols. Each experiment was performed in triplicate.+/PI−) and late (Annexin V+/PI+) apoptotic cells. All experiments were done in triplicate.sh-circZFR-infected or sh-NC-transduced cell lines were transfected with or without anti-NC or anti-miR-578, and MCF7 and BT-549 cell lines were carried out the indicated transfections, followed by the incubation for 24 h at 37 °C. Cell viability assay was done using the Cell Counting Kit-8  assay as per the manufacturer’s guidance. Cell colony formation was tested using standard colony formation protocols as previously reported . Cell apCell migration and invasion were detected by the transwell assay using modified Boyden chambers in 24-transwell plates . Chambers of cell invasion assays consisted of Matrigel-precoated membrane inserts (Corning). After transfection, BC cell lines were seeded into the top chamber of a 24-transwell insert, and medium containing 15% FCS was used as a chemoattractant in the lower chamber. 24 h later, the well was stained with 1% crystal violet (Solarbio), and the number of cells that had migrated or invaded to the basal side of the membrane was counted under a microscope  at 100× magnification. Each experiment was performed in triplicate.The Colorimetric Glucose Uptake Assay Kit, L-Lactate Assay Kit and ATP Assay Kit  were used to determine the levels of glucose uptake, lactate product and ATP, according to the recommendations of manufacturers. Briefly, the lysates of transfected cells were prepared using the Assay buffer and incubated with standard protocols for the indicated time point, followed by the measurement of the absorbance with a microplate reader (Invitrogen) at OD 412 nm for glucose uptake, 450 nm for lactate product and 570 nm for ATP level. All assays were done in triplicate.6 sh-NC-infected or sh-circZFR-transduced BT-549 cell line was subcutaneously injected into the dorsal flank of the mice. One week later, tumor size was measured every week with callipers and tumor volume was calculated using the following formula: volume = 0.5 × length × (width)2. All mice were sacrificed at 35 days after implantation, and tumor tissues were collected for weight.The xenograft models were constructed to assess the role of circZFR on tumor growth in vivo. Animal studies were implemented in accordance with a protocol approved by the Ethics Committee on Animal Use and Care of the First Affiliated Hospital of Zhengzhou University. Twelve 6–8 weeks BALB/c female mice  were used and randomly divided into two groups (n = 6 per group): sh-NC and sh-circZFR. Approximately 5 × 10https://circinteractome.nia.nih.gov/index.html) and CircBANK (http://www.circbank.cn/searchCirc.html). The putative targets of miR-578 were predicted by TargetScan v.7 online software (http://www.targetscan.org/vert_71/).Analysis for the targeted miRNAs of circZFR was performed using the online web CircInteractome  to construct corresponding wild-type reporters (circZFR-WT and HIF1A-3′UTR-WT). The TaKaRa MutanBest Kit was used to construct the corresponding mutations (circZFR-MUT and HIF1A-3′UTR-MUT) as per the insturctions of manufacturers . Each reporter construct (200 ng) and 30 nM of miR-578 mimic or miR-NC mimic were cotransfected into BC cell lines. Cell line extracts were prepared with RIPA buffer (TaKaRa) 24 h post-transfection, and the ratio of Renilla to firefly luciferase was detected using the Promega Dual-luciferase Assay. In RNA pulldown assays, cell lysates were incubated with the biotin-labeled miR-578 mimic  or nontarget control sequence  for 4 h at 4 °C before adding to the streptavidin beads (Sigma-Aldrich) for 2 h. The beads were collected, and total RNA was extracted for circZFR quantification. Each experiment was performed in triplicate.Western blot was used to determine the expression of HIF1A using standard protocols. The preparation of cell line extracts was done using RIPA buffer with proteinase inhibitors . Proteins (50 μg) were resolved on a 10% SDS–polyacrylamide gel, electrophoretically blotted onto nitrocellulose membranes  and probed with antibody against HIF1A  or GAPDH . Following the incubation with horseradish peroxidase-coupled IgG secondary antibody , the signals were visualized by Cheniluminescence  as recommended by the manufacturers. All experiments were done in triplicate.t test, Mann–Whitney U test or analysis of variance (ANOVA) with SPSS version 19.0 software . For survival analysis, the Kaplan–Meier survival curve and log-rank test were used. Correlations among circZFR, miR-578 and HIF1A expression levels in BC tissues were determined by the Spearman correlation test. All tests were considered statistically significant at p-value < 0.05.Data were shown as the mean ± standard deviation from at least three independent assays. Pairwise comparisons were done using a two-tailed Student’s As demonstrated by qRT-PCR, circZFR was significantly up-regulated in BC tissues and cell lines compared with their counterparts Fig. a, b. To To study the biological role of circZFR in BC progression, the loss-of-function experiments were carried out using shRNAs against circZFR . In contrast to the negative control, the transfection of the three shRNAs prominently reduced circZFR expression in both MCF7 and BT-549 cell lines  was identified within the 3′UTR of HIF1A Fig. a. When wWe then examine whether circZFR influenced HIF1A expression in BC cell lines. As expected, in comparison to their counterparts, HIF1A expression was remarkably down-regulated by circZFR silencing at both mRNA and protein levels in the two BC cell lines, and this effect was significantly abrogated by anti-miR-578 introduction  and BT-549 (B) cells.Additional file 2: Supplement material 2. The detailed quantification of the western blot analysis.