Anti-colon Cancer Mechanism Of Plasmodium Infection Through Mitochondria-related Pathway In Mice

Background: As a common malignancy,colon cancer with a high morbidity and mortality rate is also characterized by easy recurrence,metastasis,and poor prognosis.Therefore,the search for new treatments to inhibit the growth of colon cancer,reduce its mortality,and improve the survival quality in patients,is a goal that researchers have been pursuing.Plasmodium is one of the most relevant parasites to humans,and researchers used malariotherapy to treat patients with advanced syphilis back in the last century.In animal models,Plasmodium infection had antitumor effects,including lung cancer,melanoma,breast cancer,hepatocellular carcinoma,and leukaemia.However,there are no studies that have demonstrated the inhibitory effect of Plasmodium infection on colon cancer.At present,studies on the mechanism of antitumor effects of Plasmodium infection mainly have focused on immune mechanism,but the changes in tumor cells caused by Plasmodium infection have not received sufficient attention.Sustaining proliferative signaling and resistance to cell death are crucial hallmarks of cancer cells,and these features are essential for tumorigenesis and malignant progression.Previous studies have shown that some parasites,including Trichinella,Toxoplasma,and Schistosoma,and their proteins can promote tumor cells death through the mechanism of inducing apoptosis,thus exerting their antitumor effects.It is not clear whether Plasmodium infection inhibits colon cancer by inducing apoptosis.As we all know,mitochondria are fundamental for the growth and proliferation of cancer cells,and the imbalance in mitochondria metabolism or increased resistance to apoptosis is an important factor in promoting cancer development.The maintenance of the steady-state in the intracellular environment depends on the regulation of both mitochondrial biogenesis and mitophagy,which determine the different fates of cells.A growing number of studies have confirmed the importance of mitophagy and apoptosis in driving cancer cells death,which has become crucial for cancer therapy.Therefore,it remains unclear whether the anti-tumor effects of Plasmodium infection related to mitochondrial biogenesis,mitophagy,and mitochondrial apoptosis.Objective: This study was conducted in a murine colon cancer model,aiming to investigate the effects of Plasmodium infection on tumor growth and cell proliferation of colon cancer in mice,as well as the mechanism of tumor suppression via mitochondrial biogenesis,mitophagy,and mitochondrial apoptosis.Methods: Ten BALB/c mice were injected subcutaneously with CT26.WT cells to establish the murine colon cancer model.From the day of tumor emergence（on the 6th day）,the tumor-bearing mice were randomly divided into the experimental group（CT26.WT+P.y）and the control group（CT26.WT）,which were injected intraperitoneally with P.yoelii 17XNL-infected erythrocytes or normal mouse erythrocytes,respectively.The growth of tumor in colon cancer-bearing mice was observed dynamically,and on the 24 th day after tumor cells inoculation,the tumor-bearing mice were sacrificed to harvest tumors,weigh and photograph for subsequent analysis.The expression of Ki67,the critical indicator of cell proliferation,was detected by immunohistochemistry to determine the effect of Plasmodium infection on colon cancer cells proliferation in mice.TUNEL staining was applied to examine apoptosis,and western blot and immunohistochemistry,respectively,was applied to examine the expression of proteins in mitochondrial apoptosis pathway,including Bax,Bcl-2,Caspase-9,Caspase-3,and Cleaved Caspase-3.We used Transmission electron microscopy（TEM）to observe the ultrastructural change of colon cancer cells in mice after Plasmodium infection.JC-1 staining was used to detect the effect of Plasmodium infection on the mitochondrial membrane potential in murine colon cancer cells.Western blot was used to detect the expression of PINK1/Parkin proteins,a key pathway of mitophagy,and the expression of PGC-1α protein,a core protein of mitochondrial biogenesis.The effect of Plasmodium infection on the expression of proteins related to MDM2/p53 pathway was detected by western blot and immunohistochemistry.Results: 1.In the CT26.WT+P.y group,tumor growth was significantly slower than the CT26.WT group.And the sizes（P<0.001）and weights（P<0.001）of tumors were significantly reduced in the CT26.WT+P.y group than the CT26.WT group.Plasmodium infection decreased the expression of Ki67 compared with the control group（P<0.0001）,thereby inhibiting cell proliferation in colon cancer-bearing mice.2.In colon cancer-bearing mice,Plasmodium infection could promote mitochondrialapoptosis,as evidenced by the increased proportion of TUNEL-positive cells（P<0.0001）,the up-regulated expression of Bax,Caspase-9,Caspase-3,and Cleaved Caspase-3 proteins（P<0.0001,P<0.001,P<0.01,P<0.0001）（P<0.0001,P<0.0001,P<0.0001,P<0.0001）,and the down-regulated expression of Bcl-2 protein（P<0.01,P<0.0001）.3.Plasmodium infection could change the ultrastructural of the colon cancer cells,mainly by the destroyed cell nuclei,swollen mitochondria,missing cristae,and the decreased number of autolysosomes,resulting in disruption of mitochondrial integrity,leading to ultrastructural changes of mitochondria.4.Plasmodium infection markedly reduced the level of mitochondrial membrane potential（P<0.01）.5.Plasmodium infection attenuated mitophagy in colon cancer cells by reducing the expression of PINK1（P<0.0001）and Parkin（P<0.001）proteins.6.Plasmodium infection inhibited mitochondrial biogenesis in colon cancer cells by reducing the expression of PGC-1α（P<0.0001）protein.7.Plasmodium infection could up-relate MDM2/p53 pathway by increasing both p53（P<0.001）（P<0.0001）and MDM2（P<0.001）（P<0.0001）proteins expression.Conclusions: Plasmodium infection can inhibit mitophagy and mitochondrial biogenesis in colon cancer cells,leading to impairment of mitochondrial homeostasis and function,which in turn inhibits tumor cells proliferation and promotes mitochondrial apoptosis to inhibit tumor growth,exerting anti-colon cancer effects in mice,above which may relate to the regulation of the MDM2/p53 pathway by Plasmodium infection.