Study On The Anti-Tumor Mechanism Of The Main Active Components From Sea Cucumber

Objective:To interrogate the targeted inhibitory effect of sea cucumber（Holothuria Leucospilota）protein on tumor cell lines,and to study the inhibitory effect of sea cucumber（Cucumaria frondosa）triterpene glycoside Frondoside A on human bladder cancer cells,and to explore the feasibility and mechanism of synergistic anti-tumor effect of Frondoside A and non-methylated Cp G oligodeoxynucleotides（ODN）,providing new candidate drugs for future cancer treatment and promoting the industrialization of sea cucumber.Methods:1.To investigate the anti-tumor effect of Holothuria leucospilota protein on three tumor cell lines（i.e.,Hep G2,A549,Panc02）and three normal cell lines（NIH-3T3,Ha Ca T,16HBE）.The cell viabilities of six cell lines with Holothuria leucospilota protein treatments were examined by Cell Counting Kit-8（CCK-8）assay and the concentrations of IC₅₀were determined for subsequent experiments.The cells treated by the IC₅₀concentrations of Holothuria leucospilota protein,apoptosis and cell cycle distribution were detected by flow cytometry.The changes of cell morphology were observed by fluorescence microscope and the inhibitory effect of Holothuria leucospilota protein on cell migration was evaluated by wound healing assay.2.To study the anti-tumor effect of sea cucumber triterpene glycoside Frondoside A on human bladder cancer cells.This part mainly investigated the the cell viability,the ability of cell migration and morphological changes of bladder cancer cells with the increasing concentrations of Frondoside A.Flow cytometry assay was performed to analyze apoptosis and cell cycle distribution in bladder cancer cells UM-UC-3 treated with various concentrations of Frondoside A.3.According to the effective concentrations of the sea cucumber triterpenes glycoside Frondoside A,we determine its combination mode with different concentrations of Cp G-ODN and explore the anti-tumor effect of combination treatments.The specific contents are as follows:the viabilities of human bladder cancer cells UM-UC-3 with Frondoside A alone,Cp G-ODN alone and in combination with Frondoside A and Cp G-ODN treatments was detected by CCK-8 assay.Wound healing assay was used to detect cell migration ability,while morphological changes were observed by fluorescence microscope.Apoptosis and cell cycle phase were also measured by flow cytometry.The relative gene（TP53,Bax,Bcl-2,Caspase-3 and CDK1A）expression were tested by RT-q PCR in UM-UC-3 cells after treatments of Frondoside A alone,Cp G-ODN alone or Frondoside A combined with Cp G-ODN,and explored the potential molecular mechanism of anti-tumor effect.In vivo experiments,athymic nude mice were subcutaneously injected into the right flank with human bladder cancer cells UM-UC-3 to establish a xenograft model.After tumors reached the size of approximately 70～100 mm³,tumor-bearing mice were randomly grouped into different treatment groups.Drug-intervention treatment was performed,and the general state of health of nude mice in each group was observed and recorded.The body weights and tumor volumes of nude mice were monitored each 2 days.The experiment was terminated at 14days after intervention,and the transplanted tumor tissue was stripped.The tumor tissues were fixed with 4%paraformaldehyde,paraffin embedded,stained with hematoxylin-eosin（HE）and evaluated by tumor histopathology.Results:1.The data showed that Holothuria leucospilota protein decreased the cell viabilities of Hep G2,A549,Ha Ca T,16HBE in a concentration-dependent manner,while Panc02 and NIH-3T3 in a time-and concentration-dependent manner.We also found that the inhibitory effect of Holothuria leucospilota protein（≥10μg/m L）on cell viability is near or even superior to EPI,a clinical chemotherapeuticagent.In addition,our data also demonstrated that Holothuria leucospilota protein significantly affected the cell cycle and induced apoptosis in the three cancer cell lines investigated;in comparison,it showed no effects on the normal cell lines（i.e.,NIH-3T3,Ha Ca T and 16HBE）.In conclusion,Holothuria leucospilota protein targeted affect the cancer cell cycles and induce cancer cell apoptosis,and its superiority to inhibit cancer cell migration compared with EPI,shows the potential as a promising anti-cancer drug.2.Sea cucumber triterpene glycoside Frondoside A inhibited cell proliferation of human bladder cancer cell line UM-UC-3 in a concentration-dependent manner.Frondoside A mainly arrested sub-G1phase,exhibited a decrease in the proportion of cells in G0/G1 and a concomitant increase in the proportion of cells in G2/M and S phases thereby inducing apoptosis.It also affected the morphological changes of cells in a concentration-dependent manner,which is characterized by shrinkage of both nucleus（pyknosis）and cytoplasm,chromatin condensation（karyorhexis）,nuclear fragmentation,and the formation of apoptotic bodies.After treated with Frondoside A on UM-UC-3 cells,the effects were similar to that of positive control EPI.3.The proliferation and migration of human bladder cancer cells with Frondoside A or Cp G-ODN treatment were inhibited.When Frondoside A and Cp G-ODN used in combination,the abilities of these compounds to inhibit cell viability and migration were dramatically enhanced,having a synergistic effect.Compared with the blank control,Frondoside A combined with Cp G-ODN could induce apoptosis and affect the distribution of cell cycle,and sea cucumber triterpene glycoside Frondoside A plays a leading role.The positive control EPI promotes apoptosis in bladder cancer cells by activating the TP53 and Caspase-3pathways,while Frondoside A induces apoptosis through the expression of the anti-apoptotic Bcl-2（P<0.05）.And other pro-apoptosis genes（TP53,Bax and Caspase-3）were up-regulated,but there was no statistical significance.These results suggest that the signaling pathway that regulates Frondoside A mediated apoptosis differs from that induced by EPI.The experimental results of human bladder cancer xenograft in nude mice showed that the tumor suppression rates were 58.69%,45.93%,53.77%and 64.63%in EPI group,Frondoside A group,Cp G-ODN group and Frondoside A+Cp G-ODN group respectively,exhibiting a potent tumor growth retardation.The results confirmed that Frondoside A+Cp G-ODN could significantly enhance the tumor inhibition effect,having synergistic anti-tumor effect.There were little adverse effects on the survival status and body weight of nude mice in Frondoside A,Cp G-ODN and Frondoside A+Cp G-ODN groups,while the body weight of nude mice decreased significantly after EPI treatment.From tumor pathological sections,compared with the blank control group,cancer cells in other treatment groups exhibited relatively regular,sparse arrangement and wide cell space.Therefore,we speculated that Frondoside A plays an anti-tumor role in inhibiting tumor cell proliferation and promoting apoptotic of tumor cells.Conclusion:Sea cucumber（Holothuria leucospilota）protein showed potential anti-tumor activity toward Hep G2,A549,and Panc02 cells and were more effective in reducing cell viability and migration.The targeted effects of Holothuria leucospilota protein on tumor cells,including induction of apoptosis,suppression of metastasis on tumor cells,as well as the effect of cell cycle arrest make Holothuria leucospilota protein a promising candidate for the treatment and prevention of human cancers.Sea cucumber（Cucumaria frondosa）triterpene glycoside Frondoside A alone or in combination with Cp G-ODN exhibited superior anti-tumor activities in vitro and in vivo.The combination of Frondoside A and Cp G-ODN has a synergistic anti-tumor effect on bladder cancer with safety,feasibility and little toxic side effects.Taken together,this study indicates that it is feasible to develop low toxicity and effective anti-tumor drugs from marine organisms.