Anti-tumoral Effects Of Triptolide On Human Colon Adenocarcinoma Cell Line SW480 In Vitro And In Vivo

Triptolide(TPL), a diterpene triepoxide, is an active component of extracts derived from the medicinal plant Tripterygium wilfordii Hook F. (TWHF), it has anti-inflammatory,anti-fertility and immunosuppressive properties et al. Several reports have indicated that TPL can inhibit the proliferation of cancer cells in vitro and reduce the growth of tumors in vivo and sensitize them to chemotherapy. TPL dose has one major drawback as an antitumor agent—its toxicity, which is the major obstacles of its application in clinical therapies. Our former studies had shown that TPL inhibits the growth of human colon carcinoma cells in nude mice model, which is more effective than any other major chemotherapeutic agents for treating colorectal carcinoma. Combined treatments with several chemotherapy regimens is often used for treating enteric solid tumors. In this study, we mainly involved in investigating TPL and its anti-tumoral mechanisms, further studied the combination of TPL and other chemotherapeutic agents in treatment of digestive cancers thus reduces TPL doses for its best pharmaceutical effect. Although TPL is known to activate a number of pathways within the cell, its mechanism of action has not been well elucidated. Therefore, the aims of this study were to explore the anti-tumoral mechanism of TPL alone or in combination with other chemotherapeutic drugs.The anti-tumoral effects of TPL on SW480 cell growth arrestIn this part, we mainly study the effects of TPL on SW480 cells growth and apoptosis,topoisomerase,tubulin,HSP70 expression and others signaling molecules. Methods: 1,MTT assay was used to observe the cell growth inhibition; 2,Colony assay was to observe the cell colony forming units (CFUs); 3,AO/EB fluorescent staining was to detect the cell apoptotic rate; 4,The activities of TOPO I and TOPO II was measured by TOPO I and TOPO II mediated supercoiled pBR322 relaxation; 5, The effect of TPL on polymerization of tubulin isolated from porcine brain was determined with turbidimetry. 6,The effects of TPL on caspase-3 and caspase-9 activities were analyzed by ultraviolet spectrophotometer; 7,RT-PCR was used to evaluate the gene expression of HSP70 mRNA; 8,Western blot was to observe the effect of TPL on HSP70 and other signaling molecules which are relevant with cell growth and apoptosis; 9,The highly sensitive AO (acridine orange)-relocation method and AO-uptake method were used to detect the effect of TPL on lysosome integrity; 10,Pretreatment with SB203580 reflected TPL-induced growth arrest and apoptosis in SW480 cells.The results showed as follow: 1,TPL inhibited SW480 cell growth with time- and dose-dependent manners. IC50 values was 14.14ng/ml after 48h of TPL exposure; 2,TPL inhibited SW480 CFUs and showed dose-dependent manner; 3,TPL induced SW480 cell apoptosis and showed dose-dependent manner; 4,TPL had no effect on activity of TOPO I, only when the concentration was as higher as 400μg/ml,TPL could inhibit TOPO II enzyme activity of SW480; 5,TPL (0.1~10μg/ml) had no suppression on tubulin polymerization, however, it improved the tubulin polymerization to some degree, but seems not so significant; 6,TPL increased caspase-3 and caspase-9 activities; 7,TPL down-regulated HSP70 mRNA and protein expression at a time-and dose-dependent manners; 8,TPL down-regulated P53,c-myc,Bcl-2,p-Erk1/2 and up-regulated Bax,p-p38 protein levels but had no effect on Erk1/2 protein content; 9,Lysosomes undergo a permeabilization process in TPL-treated SW480 cell; 10,SB203580 partly blocked TPL-induced SW480 growth arrest and apoptosis.The anti-tumoral effects of the combination of TPL and OXA on SW480 cell growth arrestOne of the main reasons of chemotherapeutic resistance in colonic cancer treatment is the production of HSP70 induced by the drugs itself. As shown in the above study,TPL downregulated HSP70 expression in SW480 cells,suggested that TPL may have synergetic effect combined with OXA (Oxaliplatin). Therefore, we studied the SW480 cell proliferation and apoptosis treated by TPL and OXA;To study the effect of HSP70 and other signaling molecules expression treated by TPL and OXA; Methods: 1,MTT assay was used to access the cell growth arrest; 2,Colony assay was to observe the CFUs; 3,Flow cytometry (FCM) was used to detect the penetration on the expression of annexin V/PI; 4,The activities of caspase-3 and caspase-9 were analyzed by ultraviolet spectrophotometer; 5,Western blot was used to observe the effects of TPL and OXA on expression of HSP70 and the signaling molecules which are relevant with cell growth and apoptosis; 6,Pretreatment with SB203580 effects TPL combination with OXA-induced growth arrest and apoptosis in SW480 cell.The results showed: 1,TPL synergistically inhibited SW480 cell growth, the combined index CI<1.1 for 48 h; 2,TPL enhanced OXA to inhibit the colony forming at a dose-dependent manner; 3,Combination of TPL and OXA synergistically induced SW480 cell apoptosis; 4,TPL enhanced OXA to increase caspase-3 and caspase-9 activities; 5,OXA up-regulate HSP70 content, but TPL enhanced OXA to down-regulate HSP70 protein levels; 6,TPL enhanced OXA to down-regulate p53 expression and up-regulate Bax,p-p38 protein levels; 7,TPL and OXA had no effect on p-Erk1/2,Erk1/2; 8,SB203580 partly blocked TPL combined with OXA -induced SW480 growth arrest and apoptosis.The effect of TPL combined with OXA on SW480 cell xenograft in nude miceSince TPL enhanced OXA to inhibit SW480 cell growth and induce cell apoptosis in vitro as shown in the second part, we were interested if the both could take the same effect in SW480 nude mice and what's the possible mechanisms. Methods: 1,Mice were inoculated subcutaneously with SW480 cell; 2,Mice bearing growing tumors were randomly divided into four groups (with 6 mices/group): 2% propylene glycol saline(NS) control group, TPL 0.1mg/kg group, OXA 5mg/kg group, and TPL+OXA combination group. TPL was administered via injection into the vena caudalis every two days for seven times. Mice were given i.p. injections of OXA every three days for five times. At the end of time points, the mice were killed, and the tumor xenografts were removed and measured; 3,Blood samples obtained from mice were used to detect the blood cell counting and the biochemical markers of liver and renal injury; 4,H&E staining was used to study the pathological change of tumor tissue after treated with TPL or/and OXA ; 5,Immunohistochemistry was used to investigate the HSP70 expression in tumor tissue after treated with TPL or/and OXA; 6,The protein was extracted from the tumor tissues treated with TPL or/and OXA, Western blot was used to analyze HSP70,p53 and Bax expression.The results showed: 1,The tumor inhibition rate was 48.63% at low concentration of 0.1mg/kg of TPL (compared to the control group, p<0.01). When mice were treated with TPL combined with OXA, the tumor inhibitory rate was 59.6% (compared to the control group, p<0.01), whereas the inhibitory rate treated with OXA alone was 9.66%; 2,The results of the blood cell counting and the biochemical markers of liver and renal injury indicated that there was no difference among the combined groups, TPL group and OXA group (compared to the control group, p>0.05); 3,The results of immunohistochemistry showed decreased HSP70 expression in the combined group and TPL group, compared with control group and OXA group;HSP70 expression at the combined group was lower than TPL group; 4,The results of Western blot showed HSP70 protein levels of the combined group and TPL group were lower than control group, however the levels of OXA group was higher than control group.P53 expression decreased at all drug-giving groups while Bax expression of drug-giving groups was higher than the control.CONCLUSIONS:1. TPL inhibits SW480 cell growth with time- and dose-dependent manners. IC50 is 14.14ng/ml after 48 h of TPL exposure; TPL at concentrations of 8 to 100 ng/ml for 48 h induces SW480 cell apoptosis and showed dose-dependent manner;2. TPL at concentration of 400μg/ml inhibited activity of TOPO II , but had no effect on TOPO I;3. TPL (0.1~10μg/ml) had no suppression on tubulin polymerization, however, it improved the tubulin polymerization to some degree, but seems not so significant;4. TPL inhibits SW480 cells growth and induces cell apoptosis, this may be correlated with the down-regulation of HSP70,inhibition of p53,Bcl-2,p-Erk1/2,c-myc expression,increase of caspase-3 and caspase-9 activities,up-regulation of Bax and p-38 MAPK contents and destruction of lysosomes;5. The specific p38 MAP kinase inhibitor SB203580 reverses TPL-induced SW480 cell growth and apoptosis partly, which indicates that p38 activation is important in TPL-induced cell apoptosis;6. TPL synergizes OXA to inhibit SW480 cell growth and induce cell apoptosis, this may be correlated with the down-regulation of HSP70,inhibition of p53 expression,up-regulation of Bax and p-38 MAPK contents;7. The low concentration of TPL can inhibit xenograft growth of SW480 cells in vivo, and synergize OXA to inhibit xenograft growth of SW480 cells. No increased toxicity was shown while OXA combined with TPL in nude mice important organs. The anti-tumoral mechanism of TPL combination with OXA may be correlated with the down-regulation of HSP70,inhibition of p53 expression,up-regulation of Bax.