| Objective:To research the effects of Ginsenoside Rh2(G-Rh2) on cell proliferation, apoptosis, angiogenesis and immune regulation in T lymphocytes non-Hodgkin’s lymphoma (T cell non-Hodgkin lymphoma, T-NHL) cell line Hut-78and the antitumor function of proteasome inhibitor bortezomib (BTZ) and fludarabine (FLU) using alone or together in T-NHL in vitro.To find the effects of Ginsenoside Rh2combined with BTZ and FLU in T-NHL and its microenvironment. Demonstrating if the G-Rh2can enhance the chemotherapy sensitivity of T-NHL and searching for the molecular mechanism of the relative Signaling pathway,for providing the new treatment ideas and theoretical basis for the therapy of T-NHLMethods:1. The half maximal inhibitory concentration(IC50) of G-Rh2、BTZ and FLU was estimated by MTT to acquire the optimal concentration for the following experiment.2. Using Flow cytometry to find the effects on cell cycle when G-Rh2、BTZ and FLU were used alone or combined in HUT-78cell line.3. Detecting by flow cytometry to test the apoptosis when G-Rh2、BTZ and FLU were used alone or combined in HUT-78cell line.4. when G-Rh2、BTZ and FLU were used alone or combined,the protein level of cleaved caspase-3,Bcl-XL, Bax and P65in HUT-78cells was determined by Western blot.Results:1. MTT experiment shows:BTZ and FLU had a strong effect on inhibiting the cell proliferation when they were used alone and combined,especially for combination; The function of G-Rh2single drug acting on the inhibition of cell proliferation in HUT-78cell line was not obvious,but when it was combined with BTZ, FLU chemotherapy drugs could enhance the inbibitional effect.2. Flow cytometry test on cell cycle:G-Rh2had no specific cell cycle phase in HUT-78cell line, but FLU made the cell cycle arrest in the G0-G1phase and BTZ blocked it in the S phase.3. Flow cytometry test on apoptosis:All of the G-Rh2, BTZ and FLU could promote apoptosis, and the effect is more obvious when they are combined. 4. Western blot experiments:G-Rh2, BTZ and FLU monotherapy and combination could promote the expression of the pro-apoptotic proteins and inhibit anti-apoptotic protein expression in HUT-78cell line, and G-Rh2can promote the pro-apoptotic function of the chemotherapy.5. Western blot experiments:G-Rh2, BTZ and FLU inhibited the activation of NF-kappa B pathway, and when the triple drugs are combined this effect is strongest.Conclusions:1. Both FLU and BTZ had time and dose dependent manner,especially for the FLU; the function that G-Rh2inhibited cell proliferation was not obvious, but G-Rh2combined with chemotherapy could significantly improve the sensitivity of tumor cells to the chemotherapy drugs; FLU+BTZ synergistic effect was not obvious, but when they were combined with G-Rh2,the synergy significantly strengthened.2. Cell cycle:FLU promoted tumor cells arrested in the G0/G1phase, cells were in the dormant phase and cell proliferation slowed down; BTZ blocked cell cycle in S phase, cells were in DNA synthesis phase; Drugs acting on the cell cycle showed a dose-dependent manner; G-Rh2was cell cycle non-dependent drug.3. Apoptosis:FLU acting on cell proliferation inhibition was stronger than the induction of apoptosis; BTZ induced apoptosis effect is more prominent; G-Rh2single-drug played a weak role in inducing apoptosis, however, when it combined with chemotherapy, cell apoptosis was significantly increased.4. According to the results of Western blot:When the sample amounts were equal, both single-drug and drugs combination could strengthen the cell apoptosis, especially for the combination,and the function of three drugs combination was the most obvious, the reason of this result may be related to combination inhibition of NF-κB pathways; Use the chemotherapy drugs in different order and extract the proteins in Western blot, results showed BTZ followed by FLU induces a higher apoptosis rate, this may be related to the cell cycle state and the features of apoptosis when the drugs used in different schedule. |
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