The Mechanism Study And Effects Of Bisphosphonate CP On The Growth Of Gastric Cancer Cells

Objectives: Gastric cancer is one of the most common malignant tumorin the world.In recent years the incidence of gastric cancer is a downwardtrend, but its prognosis no obvious change.The five-year survival rate is stillless than24%in the worldwide. Chemotherapy is a major treatment means oflocal advanced and advanced gastric cancer. Over the years, gastric cancerchemotherapy seems to enter the stage of "bottleneck". Fluorouracil andplatinum drugs still is the foundation of gastric cancer chemotherapy, if jointanthracycline compounds or taxanes is controversial. Therefore to reduce themortality and prolong survival in patients with gastric cancer, to explore lowtoxicity and effective new drugs antagonist gastric cancer is the hot spots ofthe current research.Bisphosphonates drugs commonly used in the treatment of benign andmalignant bone resorption disorder. Preclinical studies have shown that atpresent, bisphosphonates have direct and indirect antitumor effect. Clinicalstudies have confirmed tumor patients with bone metastasis appliedbisphosphonates can make the patient obtain significantly benefit from it. Thedirect anti-tumor effect of bisphosphonates has been confirmed in multiplemyeloma, prostate cancer, breast cancer, and pancreatic cancer, its antitumormechanism mainly through inhibiting tumor growth and induce apoptosis.Due to structural differences, different bisphosphonates anti-tumor effect isalso different, the results showed that the bisphosphonates restrain cancer cellin vitro proliferation effect intensity is determined by its side chain groups.Bisphosphonates with complex side chain groups have the stronger inhibitioneffect than simple one. The intracellular signaling pathways ofbisphosphonates is not entirely clear. Up to now, few works focused on theproliferation and apoptosis induced by bisphosphonate CP in gastric cancer cells has not yet been reported.The extracellular-signa1regulated kinase (ERK1/2) is widely exists ineukaryotic cells of serine/threonine protein kinase, is a family members ofmitogen activated protein kinase (MAPK). From the perspective of the signaltransduction, ERK1/2may activate substrate proteins in the cytoplasm andnucleus, cause the expression of specific proteins or activation, regulation ofcell proliferation, differentiation, apoptosis, etc. ERK1/2signaling pathwaysplay the role of promoting apoptosis or inhibiting apoptosis by endogenousand exogenous cell apoptosis pathway. ERK1/2signaling pathway may beinvolved in the apoptosis induced by bisphosphonate CP. The relevance ofERK1/2signal pathways and cell apoptosis induced by bisphosphonate CP ingastric cancer has not been reported.To study the new synthesis bisphosphonate drug CP, which chemicalname [2-(6-amino-purine-9-yl)-1-hydroxy-phosphine acyl ethyl] phosphonicacid (CP), on human gastric cancer SGC-7901cells. Thus the objectives of thepresent study were:1To study the effect of CP, a new bisphosphonate derivative, onproliferation and apoptosis of human gastric cancer SGC-7901cells, and toexplore its signal pathway as well.2To investigate the role of ERK1/2signal transduction pathways in theprocess of proliferation and apoptosis induced by CP.3To observe the growth inhibitory effect and side effect of CP on humangastric cancer tumor xenografts, and to elucidate the possible mechanisminvolving in these effects.Methods：1The effects of CP on gastric cancer cells growth. Human gastric cancercell lines (SGC-7901, BGC-823, MKN-45and MKN-28) and colon cancercell lines (Lo Vo, HT-29) were cultured in RPMI-1640medium containing10%fetal bovine serum. CP dissolved in0.16%sodium bicarbonate, wasadded to the medium, with a final concentration of20,40,80or160μmol/L.MTT assay was used to determine the influence of CP on proliferation of cells and calculate the half inhibitory concentration (IC50). MTT assay was used todetermine the influence of CP on proliferation of SGC-7901cells at0,1,3,612,24hours with a final concentration of10,20,40, or80μmol/L. The cellcycle distribution and apoptosis were detected by flow cytometry, furthermore,the DNA fragments of cell apoptosis was determined by enzyme-linkedimmunosorbent assay (ELISA). The expressions of Bcl-2, Bax, Bad,caspase-3,-9, PARP protein were detected by Western blot. Meanwhile, theapoptotic enzyme activity analysis of caspase-3,-9were done after the CPtreatment24hour in gastric cancer SGC-7901cell. To observe the function ofinhibiting gastric cancer cell proliferation and induce apoptosis by CP in vitro.2The effects of CP on ERK1/2pathways in gastric cancer cell. Humangastric cancer SGC-7901cell were treated with40μmol/L CP. The expressionsof ERK1/2, p-ERK1/2, p38, p-p38, JNK, p-JNK, MEK, p-MEK, Raf-1,p-Raf-1protein were detected by Western blot at0,0.5,1,3,612, and24hours. The expression of ERK1/2mRNA were detected by RT-PCR after40μmol/L CP treatment at0,6,12,24hours. Transfection experiment Cell isdivided into8group: CP (40μmol/L) group, PD98059(20μmol/L) group, CP(40μmol/L)+PD98059(20μmol/L) group, ERK siRNA (25nmol/L) group,control siRNA (25nmol/L) group, CP (40μmol/L)+control siRNA (25nmol/L)group, CP (40μmol/L)+ERK siRNA (25nmol/L) group and control group. Theproliferation activity of each group determined by MTT. FCM was used todetect sub-G1phase cell percentage in each group. Meanwhile, the apoptoticenzyme activity analysis of caspase-9were done in each group. Theexpressions of PARP, cleaved-PARP, ERK1/2, p-ERK1/2protein weredetected by Western blot.3The effect of CP on gastric cancer nude mouse xenografts growth andits mechanism. Each of12intact female athymic nude mice (BABL/c, nu/nu,4week old,15-17g) was inoculated with SGC-7901cells (2×106/site)subcutaneously on the left axillary fossa. The animals were divided into twogroups (control and CP group). The CP group received CP (200μg/kg/day),while the control group was given vehicle (0.16%sodium bicarbonate) on day 5after inoculation and repeated daily for30day. The shortest and longestdiameter of the tumor were measured with slide gaud at3-day intervals, andtumor volume (mm3) was calculated using the following standard formula:(the longest diameter)×(the shortest diameter)2/2. On day30, mice weresacrificed and all tumor tissue samples were collected and weighed. The liverand kidneys were also dissected, fixed in10%formalin, stained withhematoxylin-eosin for histological examination. The expressions of PCNAand Ki67in tumor tissue were detected by immunohistochemistry.Terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL)was used to examine cell apoptosis in xenograft tissue. The expression ofERK1/2, pERK1/2, JNK, pJNK, p38, p-p38was assessed by Western blot.Pathological routine HE staining was used to observe the change in nudemouse transplantation tumor and liver and kidney.Results：1The effects of CP on gastric cancer cells growth.1.1Cell growth inhibition were determined by MTT. With a determined byMTT method to observe the CP for different cell growth inhibition rate, theIC50of CP at48h was about40μmol/L. CP inhibited the growth of humangastric cancer cell lines (SGC-7901, BGC-823, MKN-45) in a dose-andtime-dependent manner after incubated with10-80μmol/L CP for24hours.The normal mucosa cells of stomach (GES-1) have no inhibition by CP.1.2Cell cycle distribution and apoptosis were detected by flow cytometry. CPinduced apoptosis of gastric cancer SGC-7901cells. After incubation with CPfor24h, distinct apoptosis was observed on a DNA histogram as subdiploid orpre-G1peak, pre-G1peak was especially remarkable at24hours. Cell-cycleanalysis by flow cytometry showed that CP increased the proportion of cellsin the G2/M phase and decreased the proportion in the S phase of the cellcycle. Annexin V/PI double staining FCM analysis showed that annexin Vpositive and PI negative cells, which on behalf of apoptosis, graduallyincrease with the extension of time. 1.3The DNA fragments of cell apoptosis was determined by enzyme-linkedimmunosorbent assay (ELISA). After40μmol/L CP treatment for0,6,12,24hours, the DNA fragments of cell apoptosis was determined by ELISA. Theresults showed that DNA fragments increase gradually as the extension of CPincubation time, which reflect the apoptosis cells increase gradually。1.4The effects of CP on protein expressions of Bcl-2, Bax, Bad, caspase-3,-9,PARP. After40μmol/L CP treatment for0,6,12,24hours,40μmol/L, theexpressions of Bax, Bad, cleaved caspase-3, cleaved caspase-9, PARP proteinwere increased gradually detected by Western blot. In contrast, the expressionlevel of Bcl-2protein decreased gradually.There are significant difference (P <0.05). The ratio of Bax to Bcl-2increased.1.5The activity analysis of caspase-3,-9. The apoptotic enzyme activityanalysis of caspase-3,-9were done after the40μmol/LCP treatment24hourin gastric cancer SGC-7901cell. The activity of caspase-3, caspase-9wasincreased with time dependence (P <0.05).2The effects of CP on ERK1/2pathways in gastric cancer cell.2.1CP induced ERK1/2signal pathways activated in gastric cancer cell. After40μmol/L CP treatment for0,0.5,1,3,6,12,24hours, the expressions ofp-ERK1/2, p-raf-1, p-MEK1/2protein were increased gradually detected byWestern blot. The expressions of p-38and JNK protein which stand for theother MAPK signal pathways protein. In addition,40μmol/L CP significantlyup-regulated the expression of ERK1/2mRNA.2.2PD98059and ERK1/2siRNA decreased the role of inhibition cellproliferation produced by CP. After CP (40μmol/L), PD98059(20μmol/L) andERK siRNA (25nmol/L) alone or both combined incubation gastric cancerSGC-7901cells for24h, the cell viability was enhanced in CP(40μmol/L)+PD98059(20μmol/L) group and CP (40μmol/L)+ERK1/2siRNA(25nmol/L) group than CP (40μmol/L) alone. So we can draw a conclusionthat PD98059and ERK1/2siRNA decreased the role of inhibition cellproliferation produced by CP.2.3PD98059and ERK1/2siRNA inhibited cell apoptosis induced by CP.Compared with CP alone group, the proportion of sub-G1phase cells analyzed by flow cytometry were significantly decreased in CP(40μmol/L)+PD98059(20μmol/L) group and CP (40μmol/L)+ERK1/2siRNA(25nmol/L) group.2.4CP, PD98059and ERK1/2siRNA influence the activity of caspase-9andexpression of PARP protein. The apoptotic enzyme activity analysis showedthat the caspase-9activity was decreased in CP+PD98059group andCP+ERK1/2siRNA group than CP alone group. Meanwhile, the expression ofPARP protein was decreased in CP+PD98059group and CP+ERK1/2siRNAgroup than CP alone group.2.5CP, PD98059and ERK1/2siRNA affect ERK1/2protein expression. AfterCP (40μmol/L), PD98059(20μmol/L) and ERK siRNA (25nmol/L) alone orboth combined treatment gastric cancer SGC-7901cells for24h, theERK1/2signal pathways activated induced by CP were inhibited partially byPD98059and ERK1/2siRNA.3The effect of CP on gastric cancer nude mouse xenografts growth and itsmechanism. The model of esophageal cancer xenograft in nude mice wassuccessfully established, the tumorigenic rate in nude mice injected withSGC-7901cells was100%. Intraperitoneal injection of CP significantlysuppressed tumor growth of the xenografts (P<0.01). On day9,15,21,27and30, the mean volume of the xenograft in the CP group was51±10.17mm3,254.17±38.78mm3,358.33±43.89mm3,462.5±52.22mm3and595±55.76mm3, respectively, significantly smaller than that (119.83±8.35mm3,299.66±30.81mm3,646.33±47.50mm3,1101.57±60.47mm3and1298.33±66.16mm3, correspondingly) in the control group. The tumor weight of theCP group was0.67±0.09g, significantly lower than that of the control group(1.10±0.16g)(P<0.05). Immunohistochemical detection showed that thePCNA and Ki-67expression of xenografts tissue in CP group is significantlylower than control group (P<0.05). Compared with the control group,apoptosis cells of CP group increased in xenografts tumor, both havesignificant difference (P<0.05). Compared with the control group, theexpression level of pERK1/2is higher in CP group, the expression level of JNK, pJNK, p38p-p38has no obvious change. Then observe two groups ofnude mice liver, kidney and no abnormal change.Conclusions:1CP was demonstrated to lead to a dose-and time-dependent inhibitionproliferation and inducing apoptosis in gastric cancer cells.2CP may change the cell cycle distribution, inhibit expression of antiapoptotic proteins Bcl-2and raise caspase-3,-9protein expression which caninduce apoptosis and inhibit growth in gastric cancer cell.3CP may activate ERK1/2signal pathway, increased phosphorylationprotein expression of ERK1/2.4ERK1/2inhibitor PD98059and ERK1/2siRNA abrogated partially theability of growth and apoptosis induce by CP in gastric cancer cell5Bisphosphonate CP can increase the expression of proteins involved inERK1/2signaling pathways in vitro and in vivo. ERK1/2signaling pathwaymay be involved in CP induced gastric cancer cell apoptosis.6This experiment confirmed that the bisphosphonate CP can inhibitgrowth of gastric cancer nude mice subcutaneous transplantation tumor invivo, and no obvious side effects, and provides a theoretical basis forbisphosphonate CP in the chemical treatment of gastric cancer.