Mammalian Thioredoxin Reductase As A Drug Target In Anticancer Therapy

Mammalian thioredoxin reductase(TrxR)is a selenoenzyme,its major function is catalysis NADPH-dependent reduction of the redox-active disulfide in thioredoxin (Trx).It plays an important role in multiple cellular events related to carcinogenesis including cell proliferation,apoptosis,and cell signaling.As a selenoprotein,TrxR contains a selenocysteine on its flexible C-terminal ann which is very reactive and well-accessible during catalysis,and the pKa value of selenocysteine is 5.2,making selenocysteine in TrxR molecule present in the form of -Se~- at physiological pH。Therefore,TrxR has the propensity to be conjugated with alkylating or electrophilic agents.This oxidoreductase is over-expressed in many tumor cells;recent reports indicate it is a potential molecular target of anticancer agents.It is reported that prototype compounds representative for the major classes of clinically used anticancer alkylating agents could effectively inhibit TrxR in vitro. Cyclophosphamide(CTX)is in the nitrogen mustard group of alkylating antineoplastic chemotherapeutic agents.It has no activity against cancer cells in vitro until it undergoes metabolic activation catalyzed by liver cytochrome P450 enzymes. Therefore we investigated the influence of CTX on tumor TrxR activity in vivo and we found that CTX was a potent inhibitor of TrxR.Furthermore,we found the phenomena that TrxR recovery back to normal level in CTX-treated tumors after its inactivation,which was not seen in vitro.That may be related to the resistance of tumor cells.Our results suggest utilizing pharmacologic or genetic methods to control the recovery of TrxR activity in the treatment of CTX may be a rational therapeutic strategy to overcome the CTX-resistance.We also used ascites H22 cells to investigate cancer cells response after TrxR was inhibited by CTX in vivo。The observations provide direct evidence that TrxR is essential to tumor growth,and inhibition of TrxR activity in malignant cells by CTX is tightly connected with its anticancer effect.The TrxR inhibition could thereby be considered as a crucial mechanism contributing to anticancer effect seen upon clinical use of CTX.Heart failure is a life-threatening complication of high-dose CTX chemotherapy, but little is known about its mechanism.The present study demonstrates CTX-evoked heart failure involves pronounced co-suppression of cytoplasmic TrxR(TrxR1) activity and non-protein free thiol(NPFT)level.It has been reported that TrxR1 is essential in most tissues except heart.The present study also found cardiac TrxR1 was unique in two aspects.Firstly,our previous studies showed that half inactivation of TrxR1 due to CTX or ifosfamide treatment is associated with a toxicological consequence.In contrast,in the present study,when cardiac TrxR1 activity was suppressed by 68%,no cardiac toxicity was observed.Secondly,our previous studies showed that TrxR1 activity in tumor tissue,ascitic cancer cells,bladder and kidney rapidly recovered back to normal level after being inactivated by CTX or ifosfamide, while in the present study,TrxR1 activity in heart recovered less efficiently.TrxR1 inactivation plays a role in CTX-evoked heart failure,because inactivated TrxR1 gains cytotoxic function,which would emerge under certain circumstance,such as NPFT depletion.Indeed,the present study revealed heart failure broke out when TrxR1 activity and NPFT level were pronouncedly co-suppressed by two ways,one is the high-dose CTX itself,and the other is the moderate-dose CTX plus buthionine sulfoximine,an inhibitor of glutathione synthesis.Ifosfamide is an oxazaphosphorine alkylating agent with a broad spectrum of antineoplastic activity.It is a prodrug that has first to be transformed by hepatic cytochrome P-450 enzymes.We have found that CTX potently inhibited the activity of tumor TrxR in vivo.As a structural analog of CTX,ifosfamide has not yet been studied for possible interaction with mammalian TrxR.Therefore we investigated the effect of ifosfamide on TrxR activity in mice.We demonstrated ifosfamide inhibited TrxR activity in vivo,and led to a subsequently dramatic deceleration in tumor progression.In addition,ifosfamide preferentially inhibited the activity of TrxR in vivo over other antioxidant parameters.Interestingly,the ifosfamide-treated cancer cells with decreased TrxR activity lost total tumorigenicity after being inoculated into mice.Ifosfamide could also inhibit TrxR activity in kidney,bladder and bladder. These observations provide direct evidence that inhibition of TrxR activity in malignant cells by ifosfamide is highly associated with its anticancer effect and the mechanism of ifosfamide systemic toxicity may be related to multi-organ inhibition of TrxR activity.The TrxR activity inhibition could be considered as a pivotal mechanism contributing to anticancer effect of ifosfamide.In view of the results mentioned above,the TrxR enzyme target protection may be an effective means to reduce or alleviate the tissue toxicity of anticancer agents, and then higher dose anticancer agents can be used to harvest better therapeutic effect. Amifostine is a pharmacological antioxidant used as a broad-spectrum cytoprotective agent in cancer chemotherapy and radiotherapy.FDA approval was granted in 1995 for a substantial attenuation of cumulative nephrotoxicity in patients receiving repeated doses of cisplatin(CDDP).It has been demonstrated via ex vivo experiments that CDDP inactivates TrxR,but whether CDDP at pharmacological doses inhibits TrxR activity in vivo has not been reported hitherto.Present study in mice revealed CDDP at pharmacological doses inhibited TrxR activity in both ascitic hepatoma 22 (H22)cells and kidney,leading to suppression of ascitic H22 cells growth along with nephrotoxicity.Furthermore,amifostine deterred CDDP-generated inhibition of renal TrxR activity but did not interfere with TrxR inactivation by CDDP in ascitic H22 cells.The large enhancement of CDDP cure rate in H22 ascites model by using amifostine is,at least in part,ascribed to its selective modulation on TrxR.