most patients have advanced disease when they are diagnosed , and most experience relapse and metastasis after treatment .
the prognosis of advanced gastric cancer is very poor ; therefore , systemic multidisciplinary treatment of gastric cancer is particularly important , and chemotherapy plays a critical role in advanced gastric cancer treatment .
however , the response rate of cddp treatment against advanced gastric cancer and the survival of patients remain unsatisfactory .
also , chemotherapy is often limited by dose - related toxicity , which restricts advanced gastric cancer treatment .
thus , developing more effective and less toxic therapeutic regimens for new therapeutic agents is urgently required for sensitizing gastric cancer cells to chemotherapy to improve the prognosis .
steroidal saponins have significant bioactivities , including their antitumor , hemostatic , immunotropic , and analgesic properties .
paris saponins are the most important potential agents that can be developed for new antitumor medicines [ 213 ] . among them , paris saponin i ( psi ) has been extensively studied .
the major advantages of psi as an anticancer agent are related to its ability to inhibit tumor growth in various cancer types [ 1419 ] associated with a low level of toxicity .
recent studies have demonstrated that psi possesses pharmacological activities concerning the cytotoxic activity against many cancers with mechanism of increasing levels of bax , cytochrome c , activating caspases , and by decreasing both bcl-2 expression and specific kinase-1/2 activity [ 1419 ] .
psi may be a potential anticancer drug by inhibiting proliferation and a good radiosensitizer of gefitinib resistant nsclc cells .
however , the relationship between psi and the sensitivity of gastric cancer cells to cisplatin has not been reported .
this study was conducted to explore the possibility of using psi as an agent to sensitize cancer cells to cisplatin , with an attempt to provide new methods and insights for the management of gastric cancer .
paris saponin i ( ps i ) ( c44h70o16 ) was obtained from the zhejiang institute for food and drug control ( batch no .
the purity was greater than 99% , and it was dissolved in dimethyl sulfoxide ( dmso ) and stored at 20c .
the drugs were diluted in rpmi-1640 to achieve the final concentration used for following experiment with the final dmso concentration less than 0.25% ( v / v ) .
the rpmi-1640 medium and fetal calf serum were obtained from hyclone co. ( logan , ut ) .
the cycle test plus dna reagent kit and fitc annexin v apoptosis detection kit were obtained from bd biosciences ( nj , usa ) .
rabbit anti - rat b cell lymphoma 2 ( bcl-2 ; # 3498 ) , bcl-2-associated x protein ( bax ; # 5023 ) , caspase-3 ( # 9665 ) , and p21/waf1/cip1 ( # 2947 ) monoclonal primary antibodies at 1 : 1000 dilution ( cell signaling technology , danvers , ma ) ; anti - gapdh ( santa cruz biotechnology , inc . , dallas , tx ) .
the human gastric cancer cell line sgc-7901 was obtained from the atcc company ( manassas , va , usa ) .
cells were grown in rpmi-1640 culture medium with 10% fetal bovine serum , 100 g / ml penicillin and 100 g / ml streptomycin at 37c in a 5% co2 humidified atmosphere .
the antiproliferative effects of psi , cisplatin - alone , and cisplatin plus psi were assessed using a mtt assay .
sgc-7901 cells ( 100 l / well , 110 cells / ml ) were seeded and each group had triplicate treatments . meanwhile , a nontreated group was established as the control ( dmso concentration was 0.25% ( v / v ) ) , and then treated with different concentrations of psi ( 0.2 , 0.4 , 0.8 , 1.6 , 3.2 , 6.4 g / ml ) , or cisplatin ( 0 , 1 , 2 , 4 , 8 , 16 , 32 , 64 m ) , or a combination of cisplatin ( at the concentrations shown above ) plus psi ( 0.3 g / ml ) for 48 h. dose - dependent curves were generated .
the cytotoxic effects of tested agents were expressed as the 50% inhibiting concentration ( ic50 ) .
four groups were divided as the control group , psi group , cisplatin group and psi + cisplatin group .
the concentration of dmso in the control group was 0.25% ( v / v ) .
psi ( 0.3 g / ml ) and cisplatin ( 0 , 8,16 , 32 m ) were used to treat the cells for 48 h , and then cells were harvested and fixed with 70% ethanol which were stored overnight at 20c .
flow cytometry with a kaluza software , version 1.20 ( beckman coulter , inc . , brea , ca , usa ) was used to analyze cell cycle .
annexin v / pi method was used to analyze apoptosis with annexin v fitc apoptosis detection kit ( bd biosciences ) .
four groups were divided as the control group , psi group , cisplatin group and psi + cisplatin group .
the concentration of dmso in the control group was 0.25% ( v / v ) .
cells were treated with psi ( 0.3 g / ml ) and cisplatin ( 0 , 8 , 16 , 32 m ) , then harvested at 48 h , and stained following the kit instruction .
cells were incubated with the mixture of annexin v fitc and pi in the dark .
four groups were divided as the control group , cisplatin group and psi + cisplatin group .
the concentration of dmso in the control group was 0.25% ( v / v ) .
cells were treated with psi ( 0.3 g / ml ) and cisplatin ( 16 m ) , following treatment , cells were lysed with lysis buffer containing 50 mm tris - hcl ( ph 8.0 ) and 150 mm 1% tritonx-100 ( sigma - aldrich ) .
equal amounts of protein were separated by 10% sodium dodecyl sulfate - polyacrylamide gel electrophoresis , then transferred to nitrocellulose membranes ( thermo fisher scientific , inc . , waltham , ma , usa ) .
the membranes were incubated overnight at 4c with primary antibodies ( rabbit anti - rat bcl-2 , bax , caspase-3 and p21/waf1/cip1 monoclonal antibodies and mouse anti - rat gapdh monoclonal antibody ( 1 : 1,000 ) ) , and then washed three times with tris - buffered saline supplemented with tween-20 , prior to being incubated for 2 h at room temperature with the hrp - conjugated goat anti - rabbit igg secondary antibody ( 1 : 10,000 ) .
the membranes were visualized using an enhanced chemiluminescence system and x - ray films ( santa cruz biotechnology inc . ) .
groups were compared by one - way anova test and snk - q test considering p<0.05 as a significance level .
paris saponin i ( ps i ) ( c44h70o16 ) was obtained from the zhejiang institute for food and drug control ( batch no .
the purity was greater than 99% , and it was dissolved in dimethyl sulfoxide ( dmso ) and stored at 20c .
the drugs were diluted in rpmi-1640 to achieve the final concentration used for following experiment with the final dmso concentration less than 0.25% ( v / v ) .
the rpmi-1640 medium and fetal calf serum were obtained from hyclone co. ( logan , ut ) .
the cycle test plus dna reagent kit and fitc annexin v apoptosis detection kit were obtained from bd biosciences ( nj , usa ) .
rabbit anti - rat b cell lymphoma 2 ( bcl-2 ; # 3498 ) , bcl-2-associated x protein ( bax ; # 5023 ) , caspase-3 ( # 9665 ) , and p21/waf1/cip1 ( # 2947 ) monoclonal primary antibodies at 1 : 1000 dilution ( cell signaling technology , danvers , ma ) ; anti - gapdh ( santa cruz biotechnology , inc . , dallas , tx ) .
the human gastric cancer cell line sgc-7901 was obtained from the atcc company ( manassas , va , usa ) .
cells were grown in rpmi-1640 culture medium with 10% fetal bovine serum , 100 g / ml penicillin and 100 g / ml streptomycin at 37c in a 5% co2 humidified atmosphere .
the antiproliferative effects of psi , cisplatin - alone , and cisplatin plus psi were assessed using a mtt assay .
sgc-7901 cells ( 100 l / well , 110 cells / ml ) were seeded and each group had triplicate treatments . meanwhile , a nontreated group was established as the control ( dmso concentration was 0.25% ( v / v ) ) , and then treated with different concentrations of psi ( 0.2 , 0.4 , 0.8 , 1.6 , 3.2 , 6.4 g / ml ) , or cisplatin ( 0 , 1 , 2 , 4 , 8 , 16 , 32 , 64 m ) , or a combination of cisplatin ( at the concentrations shown above ) plus psi ( 0.3 g / ml ) for 48 h. dose - dependent curves were generated .
the cytotoxic effects of tested agents were expressed as the 50% inhibiting concentration ( ic50 ) .
four groups were divided as the control group , psi group , cisplatin group and psi + cisplatin group .
the concentration of dmso in the control group was 0.25% ( v / v ) .
psi ( 0.3 g / ml ) and cisplatin ( 0 , 8,16 , 32 m ) were used to treat the cells for 48 h , and then cells were harvested and fixed with 70% ethanol which were stored overnight at 20c .
flow cytometry with a kaluza software , version 1.20 ( beckman coulter , inc . ,
annexin v / pi method was used to analyze apoptosis with annexin v fitc apoptosis detection kit ( bd biosciences ) .
four groups were divided as the control group , psi group , cisplatin group and psi + cisplatin group .
the concentration of dmso in the control group was 0.25% ( v / v ) .
cells were treated with psi ( 0.3 g / ml ) and cisplatin ( 0 , 8 , 16 , 32 m ) , then harvested at 48 h , and stained following the kit instruction .
cells were incubated with the mixture of annexin v fitc and pi in the dark .
four groups were divided as the control group , cisplatin group and psi + cisplatin group .
the concentration of dmso in the control group was 0.25% ( v / v ) .
cells were treated with psi ( 0.3 g / ml ) and cisplatin ( 16 m ) , following treatment , cells were lysed with lysis buffer containing 50 mm tris - hcl ( ph 8.0 ) and 150 mm 1% tritonx-100 ( sigma - aldrich ) .
equal amounts of protein were separated by 10% sodium dodecyl sulfate - polyacrylamide gel electrophoresis , then transferred to nitrocellulose membranes ( thermo fisher scientific , inc . , waltham , ma , usa ) .
the membranes were incubated overnight at 4c with primary antibodies ( rabbit anti - rat bcl-2 , bax , caspase-3 and p21/waf1/cip1 monoclonal antibodies and mouse anti - rat gapdh monoclonal antibody ( 1 : 1,000 ) ) , and then washed three times with tris - buffered saline supplemented with tween-20 , prior to being incubated for 2 h at room temperature with the hrp - conjugated goat anti - rabbit igg secondary antibody ( 1 : 10,000 ) .
the membranes were visualized using an enhanced chemiluminescence system and x - ray films ( santa cruz biotechnology inc . ) .
groups were compared by one - way anova test and snk - q test considering p<0.05 as a significance level .
psi concentrations of 0.2 to 6.4 g / ml caused dose - dependent inhibition of the sgc-7901 cell growth at 48 h ( figure 1a ) .
the ic50 value of psi in sgc-7901 cell lines at 48 h was 1.12 g / ml , indicating that psi had a significant anticancer activity in gastric cancer cells in vitro .
then the cells were treated with different concentrations of cisplatin , either alone or in combination with psi ( choose ic20 , 0.3 g / ml ) at 48 h. cisplatin at concentrations of 1 to 64 m caused a dose - dependent inhibition at 48 h ( figure 1b ) .
the ic50 value of cisplatin in sgc-7901 cell lines was 30.4 m at 48 h. when cisplatin was combined with psi ( 0.3 g / ml ) , the ic50 value decreased significantly to 20.3 m ( figure 1b ) .
the results indicate that psi significantly sensitizes sgc-7901 cell lines to cisplatin - induced proliferation inhibition .
cisplatin induced g2/m phase arrest in a concentration - dependent manner at 48 h. furthermore , psi promoted cisplatin - induced g2/m phase arrest in a cisplatin concentration - dependent manner ( figure 2 ) .
the results show that psi promotes cisplatin - induced inhibition of sgc-7901 cell proliferation according to the g2/m phase arrest .
annexin v / pi double staining assay was used to evaluate the apoptosis induced by psi and cisplatin in sgc-7901 cells .
apoptosis was observed in cisplatin treated group with different concentrations , and furthermore , significant apoptosis was observed in psi plus cisplatin group compare to cisplatin treated group ( figure 3 ) .
the results reveal that psi promotes cisplatin - induced apoptosis in sgc-7901 cells at 48 h significantly in a cisplatin concentration - dependent manner . to explore the mechanism by which psi enhances the cisplatin - induced apoptosis and cell cycle arrest in sgc-7901 cells
, we examined the effects of psi and/or cisplatin on the expression of key regulators , including caspase-3 , bax , bcl-2 and p21 .
caspase-3 , bax and bcl-2 were the most important apoptosis regulators and p21 was the most important cell cycle checkpoint regulator .
cisplatin treatment decreased the level of bcl-2 and increased the levels of caspase-3 , bax , p21 , however , psi enhanced these effects ( figure 4 ) .
the result suggested that the regulation of p21 , caspase-3 , bax and bcl-2 expressions contributed to g2/m phase arrest and apoptosis induced by psi to cisplatin in sgc-7901 cells .
psi concentrations of 0.2 to 6.4 g / ml caused dose - dependent inhibition of the sgc-7901 cell growth at 48 h ( figure 1a ) .
the ic50 value of psi in sgc-7901 cell lines at 48 h was 1.12 g / ml , indicating that psi had a significant anticancer activity in gastric cancer cells in vitro .
then the cells were treated with different concentrations of cisplatin , either alone or in combination with psi ( choose ic20 , 0.3 g / ml ) at 48 h. cisplatin at concentrations of 1 to 64 m caused a dose - dependent inhibition at 48 h ( figure 1b ) .
the ic50 value of cisplatin in sgc-7901 cell lines was 30.4 m at 48 h. when cisplatin was combined with psi ( 0.3 g / ml ) , the ic50 value decreased significantly to 20.3 m ( figure 1b ) .
the results indicate that psi significantly sensitizes sgc-7901 cell lines to cisplatin - induced proliferation inhibition .
cisplatin induced g2/m phase arrest in a concentration - dependent manner at 48 h. furthermore , psi promoted cisplatin - induced g2/m phase arrest in a cisplatin concentration - dependent manner ( figure 2 ) .
the results show that psi promotes cisplatin - induced inhibition of sgc-7901 cell proliferation according to the g2/m phase arrest .
annexin v / pi double staining assay was used to evaluate the apoptosis induced by psi and cisplatin in sgc-7901 cells .
apoptosis was observed in cisplatin treated group with different concentrations , and furthermore , significant apoptosis was observed in psi plus cisplatin group compare to cisplatin treated group ( figure 3 ) .
the results reveal that psi promotes cisplatin - induced apoptosis in sgc-7901 cells at 48 h significantly in a cisplatin concentration - dependent manner .
to explore the mechanism by which psi enhances the cisplatin - induced apoptosis and cell cycle arrest in sgc-7901 cells , we examined the effects of psi and/or cisplatin on the expression of key regulators , including caspase-3 , bax , bcl-2 and p21 .
caspase-3 , bax and bcl-2 were the most important apoptosis regulators and p21 was the most important cell cycle checkpoint regulator .
cisplatin treatment decreased the level of bcl-2 and increased the levels of caspase-3 , bax , p21 , however , psi enhanced these effects ( figure 4 ) .
the result suggested that the regulation of p21 , caspase-3 , bax and bcl-2 expressions contributed to g2/m phase arrest and apoptosis induced by psi to cisplatin in sgc-7901 cells .
although new compounds have been developed to be active against transitional cell carcinoma , cisplatin remains one of the most active 
standard single - agents and the mainstay of typical combination regimens against gastric cancer [ 2226 ] . however , cisplatin is highly toxic , and its efficacy is limited owing to its adverse events .
thus , developing effective agents to sensitize cisplatin efficacy and overcome platinum resistance in gastric cancer makes a challenge .
paris saponin i , originally derived from rhizoma paridis , which is the root of chinese herbal , has been recently investigated as a new anticancer agent [ 1419 ] . in the present study
we found that the ic50 value of cisplatin in sgc-7901 cell lines was 30.4 m at 48 h. when cisplatin was combined with psi ( 0.3 g / ml ) , the ic50 value decreased significantly to 20.3 m , revealing that psi significantly sensitized sgc-7901 cell lines to cisplatin - induced proliferation inhibition .
the most important effect of chemotherapy for treating human cancers is cell proliferative inhibition according to the induction of cell apoptosis .
cisplatin is one of the most commonly used drug in treating various types of cancer .
it has shown that cisplatin can bind to dna and inhibit dna synthesis , suppress cell division and induce apoptosis .
it has been increasingly recognized that the apoptosis of tumor cells is a key indicator for measuring the effectiveness of chemotherapy , and the apoptosis is associated with the chemotherapy drugs in a time- and dose - dependent manner .
there are two major apoptosis pathways : an extrinsic pathway , which involves signal transduction through cell surface death receptors , and an intrinsic pathway , which is triggered by radiation or chemical agents .
therefore , targeting the regulation of apoptosis represents an important pharmacological strategy for the development of chemotherapeutic agents .
caspase-3 is the most important death protease in the caspase family , which play vital roles in mediating interactions through the apoptotic process , catalyzing the specific cleavage of many key proteins in the apoptosis pathway .
the bcl-2 family is composed of a series of anti - apoptotic and proapoptotic members .
bcl-2 and bax are the crucial mediators of cell apoptosis between anti - apoptosis and proapoptosis .
bcl-2/bax ratio is regarded as an indicator of apoptosis level , and a high bcl-2/bax value indicates cell resistance to apoptosis .
therefore , pharmacological manipulation of caspases and bcl-2 family is a potential therapeutic strategy . in the present study ,
apoptosis was increased by cisplatin treatment and was increased significantly by psi plus cisplatin treatment .
further , cisplatin treatment decreased the level of bcl-2 and increased the levels of caspase-3 , bax , and psi enhanced these effects of cisplatin .
thus , psi induces apoptosis , especially together with the cisplatin treatment , leading to enhanced efficacy
. the mechanism may be by the regulation of bcl-2 , bax , and caspase-3 .
psi enhanced the effect of cisplatin to alter the cycle distribution with g2/m phase arrest in gastric cancer cells .
further study showed that psi significantly increased the expression of p21 , finally resulting in cell cycle blocking of g2/m phase , which indicates that p21 is an important mediator in cell cycle progression .
psi with cisplatin can induce significant g2/m phase arrest through p21 regulation in gastric cancer cells .
psi significantly sensitizes sgc-7901 cell lines to cisplatin - induced proliferation inhibition in a cisplatin concentration - dependent manner in vitro .
the mechanism of this chemosensitivity is related to the g2/m phase arrest and apoptosis by increased caspase-3 , bax , p21 and decreased bcl-2 expression
. therefore , psi shows promise as a chemosensitizer , but this needs further investigation in vivo .
 backgrounddose - related toxicity is the major restriction of cisplatin and cisplatin - combination chemotherapy , and is a challenge for advanced gastric cancer treatment . 
 we explored the possibility of using paris saponin i as an agent to sensitize gastric cancer cells to cisplatin , and examined the underlying mechanism.material/methodsgrowth inhibition was detected by mtt assay . 
 the cell cycle and apoptosis were detected using flow cytometry and annexin v / pi staining . 
 the p21waf1/cip1 , bcl-2 , bax , and caspase-3 protein expression were detected using western blot analysis.resultsthe results revealed that psi sensitized gastric cancer cells to cisplatin , with low toxicity . 
 the ic50 value of cisplatin in sgc-7901 cell lines was decreased when combined with psi . 
 psi promoted cisplatin - induced g2/m phase arrest and apoptosis in a cisplatin concentration - dependent manner . 
 bcl-2 protein expression decreased , but bax , caspase-3 , and p21waf1/cip1 protein expression increased with psi treatment.conclusionsthe underlying mechanism of paris saponin i may be related to targeting the apoptosis pathway and cell cycle blocking , which suggests that psi is a potential therapeutic sensitizer for cisplatin in treating gastric cancer . 