Antitumor Effect And Mechanistic Study Of Compound Kushen Injection On Malignant T Cell Lymphoma

BackgroundMalignant lymphoma(ML)is a group of malignant tumors that originate from the lymphatic system,which mostly occurs in lymph nodes and/or the extranodal tissue and can be subdivided into T cell-derived lymphomas and B-cell derived lymphomas by different leukocyte differentiation antigens.T-cell lymphoma is less common clinically than B-cell lymphoma,accounting for only about 10%-15%of all lymphomas.However,due to its high incidence in Asian populations,strong individual heterogeneity,wide symptoms specificity,low treatment effective rate,and poor treatment outcomes,most of these lymphomas recur within 2-3 years after treatment and thus attract attentions of research investigators to identify the effective treatment strategy nationally and internationally.Compound Kushen Injection(CKI),considering its antitumor,analgesic,and immune-enhancing effects,is often used for maintenance treatment and adjuvant to radiotherapy or chemotherapy in advanced cancer patients.Prof.Lin Hongsheng,chief researcher of China Academy of Chinese Medical Sciences and former director of Oncology Department at Guang'anmen Hospital,China Academy of Chinese Medical Sciences,has found that CKI can be used to treat slowly progressing T-cell lymphoma,especially for patients with multiple forms of skin lesions.CKI can help alleviate symptoms and significantly improve quality of life and therefore provide an unique approach in treating lymphoma patients with slow progression or no lymph node or extranodal organ being involved except the presence of the skin lesion since there is no effective treatment modalities available for this particular type of cancer and they can only be observed.PurposeThis study set forth to determine the therapeutic effects of CKI on the biological behavior of cancer cells and relevant molecular mechanisms,including cancer cell metabolic pathways of in vitro and in vivo lymphoma cells and their relevant syngeneic mouse model,and further explored the immune regulating mechnisms in a mouse model,to provide scientific evidences to support further evaluation of CKI in future clinical trial in patients with T-cell lymphoma.Method1.Effects of CKI on the Proliferation,Apoptosis and Cell Cycle of Lymphoma Cells1.1 Effect of CKI on the proliferation of different lymphoma cell lines Human or mouse lymphoma cell lines:Hut78(human skin T-cell lymphoma cells),Loucy(human T lymphocytic leukemia cells),Jurkat(human T lymphocytic leukemia cells),Raji(human B-cell lymphoma cells)and EL4(murine T lymphocytic leukemia cell).We chose prestoblue staining method to measure the proliferation of cancer cells after comparing the sensitivity of CCK8 and prestoblue and the interference of the CKI on the CCK8 cell proliferation method.Prestoblue staining and cell counting by hemocytometer were used to verify the effects of different concentrations(0.039-5mg/ml)of CKI on the proliferation of different lymphoma cells at 24h,48h,72h and 96h after intervention.The morphological changes of the cells were also observed microscopy to examine the sensitivity of lymphoma cell lines to CKI treatment.1.2 Effect of CKI on cell cycle analysis of T-cell lymphoma Hut78 and Loucy cells PI staining was used to explore cell cycle changes of the CKI sensitive cell lines Hut78 and Loucy cells after being treated with CKI for hrs?1.3 Effect of CKI on apoptosis of T-cell lymphoma Hut78 and Loucy cells 7AAD/AnnexinV double staining was used to determine the ability of CKI on the induction of apoptotic cell death in Hut78 and Loucy cells.The percentage of the apoptotic cells was measured by flow cytometry.2.Effect of CKI on Cell Signaling Pathway in Hut78 and Loucy cells Reverse Phase Proteomic Array(RPPA)was used to determine the expression of 350 cell cycle and apoptosis regulatory proteins and other cell signaling protein in Hut78 cells 24h after intervention of different doses of CKI and cluster analysis was used to identify the potential signaling pathways.Western blotting(WB)was used to verify cancer cell signaling pathway altered by CKI in Hut78 and Loucy cells to identify the regulatory mechanism of CKI on inhibiting cell proliferation and inducing apoptosis.3.Effect of CKI on Cancer Cell Metabolism in Hut78 and Loucy Cells High-performance liquid chromatography(HPLC)and tendom mass spectrometry was used for separation and quantification of glycolysis and glutamine metabolites in CKI treated Hut78 and Loucy cells after 24 hrs of treatment.4.Antitumor Efficacy of CKI on mouse EL4 Lymphoma model4.1 Effect of CKI on survival of mouse syngeneic EL4 lymphoma modelC57BL/6J mice were used to develop an animal model of EL4 lymphoma by subcutaneously injecting the EL4 cells to the flank of the mice.The tumor-bearing mice were randomly assigned to low(2ml/kg),medium(4 mg/kg)and high doses(8ml/kg)of CKI group,methotrexate(MTX)group and saline vehicle control group.The tumor-bearing mice were treated in vivo for 14 days,and the tumor size,mouse body weight and survival time were measured.4.2 Effect of CKI on immunoregulation and metabolism of EL4 lymphoma modelEL4 cells were subcutaneously injected to C57BL/6J mice to develop an animal model of EL4 lymphoma.The tumor-bearing mice were randomly divided into the optimal dose of the CKI group(2ml/kg and 4ml/kg)and physiological saline group.The tumor-bearing mice were treated with CKI for 7 days.The size of the tumors and body weight were measured.Immune profiling(IP)was utilized to detect immune cell profile in mouse spleen and tumor tissues.LC/MS was used to test the tumor metabolites of tumor tissue.All the above were used to identify the mechanisms of CKI in vivo.Paraffin sections of mouse tumor tissues were prepared and Cleave Casepase3 IHC staining was used to detect the apoptosis of tumor tissues.Result1.CCK8 and Prestoblue were both sensitive in cell viability detection of CKI treated lymphoma cells,but the color of CKI has less influence on Prestoblue assay.So we chose Prestoblue staing method to measure the proliferation of lymphoma cells treated with CKI.Cell viability counted by hemocytometer was used to verify the effects of CKI on the proliferation of the lymphoma cell.Data obtained by Prestoblue staining showed that CKI inhibited the proliferation of all 5 lymphoma cell lines tested,among which T-cell lymphoma cell lines Hut-78,EL4,Loucy,Jurkat are more sensitive then B-cell lymphoma Raji.Hut-78 cells were most sensitive to CKI treatment followed by Loucy cells.The antiproliferative effect of CKI on these lympoma cells were time-and dose-dependent.Cell counts done with hemocytometer also showed the samiliar trend as that of Prestoblue assay.Result of cell cycle and apoptosis analysis demonstrated that that there was no obvious G0/G1,S or G2/M arrest in Hut-78 or Loucy treated with CKI.The cell populations of subG0/G1 group was elevated and this effect was dose-dependent,which indicated that CKI treatment can lead to cells undergone apoptotic or necrotic cell death which may play a critical role in tumor inhibition of CKI.7AAD/Annexin V staining showed that CKI indeed induced the apoptosis in both Hut-78 and Loucy cells.The induction of apoptosis by CKI was further supported by increased expression of cleaved caspase3 and caspase 7 in these two cancer cell lines.But there was a minor difference between this two cell line—the proportion of early apoptosis was larger in Hut-78 than that in Loucy and that of late apoptosis and necrosis showed the opposite trend and this suggests that the time of apoptosis induced by CKI is late or there are other related necrosis inducing pathways.2.Proteomic analysis result revealed that CKI significantly down regulated the proteins associated with PI3K/Akt/mTOR,NF-KB?MEK/ERK pathways in Hut78 cells.may be the potential pathways of CKI on lymphoma.WB data confirmed that CKI reduced the expression of the proteins of PI3K/Akt/mTOR pathway,such as Akt,pAkt,mTOR,and S6 proteins and upregulated AMPK and TSC1 protein expression suggesting PI3K/Akt/mTOR may play an important role in CKI elicated antiproliferative activity in both lymphoma Hut-78 and Loucy cells.suggesting which in turn regulated AMPK/TSC1/mTor/S6 pathway and thus inhibited cell proliferation or induced cell apoptosis in both cell lines.Therefore,PI3K/Akt/mTOR and AMPK/TSC1/mTor/S6 downstream may be the main mechanism of CKI's inhibiton on T-cell lymphoma.3.HLPC/MS result showed that CKI was capable of alterating the metabolism of glycosis and glutamine metabolism by reducing the levels of the lactate and glutamate in Hut78 and loucy cells.WB test demonstated that CKI could also downregulate the expression of GLS1 and LDHA which are two important enzymes involved in glycolysis and glutmaminolysis.Together,these data suggested that CKI could reduce the expression and activity of the enzymes important in glycolysis and glutamine metabolism which might lead to reduce the energy supply to the tumor and resulted in slowing down the cell growth.4.In the C57BL/6J mouse model of EL4 lymphoma,we found that CKI exerted similar antitumor activity to MTX with respect to improving survival rate and prolonging survival time.The samilar tumor inhibitory effect was seen in tumor growth rate measured by volume.Immune cell subpopulation detection showed that CKI could upregulate T cells(CD3+),among which most were Th(CD3+CD4+),Treg(CD4+FoxP3)and could also downregulate Th17(CD3+CD4+IL17)/Treg(CD4+FoxP3).This suggest that the anti-tumor effect of CKI may be associated with immune modulation and related inflammation.LC/MS test showed that CKI reduced the levels of glutamine,glutamate and lactate in the tumor tissues which was consistent with that being observed in CKI treated Hut-78 and Loucy cell lines in vitro,further suggesting that CKI can help reduce energy supply of tumor by inhibiting both glycolysis and glutamine metabolism in the tumor.Conclusion1.CKI inhibited the proliferation of lymphoma cells and T cell lymphoma was more sensitive to CKI treatment than B cell lymphoma.This was potentially achieved by induction of apoptotic cell death in these cells.2.PI3K/Akt/mTOR and AMPK could be a mechanism of CKI's inhibiton on T-cell lymphoma.3.CKI can reduce the levels of glutamine,glutamate and lactate and thus suppress glycolysis and glutamine metabolism which was demonstrated in both in vitro and in vivo settings.4.CKI can slow down tumor growth and improve survival rate and prolong survival time of EL4 lymphoma mouse model which is similar to that of MTX treatment.5.CKI can downregulate Thl7(CD3+CD4+IL17)/Treg(CD4+FoxP3),which indicates that the anti-tumor effect of CKI may lie on inhibiting cancer related inflammation.