Cancer cell expulsion of anticancer drugs through shedding of microvesicles: association with drug resistance and tumour survival

Microvesicles (MVs) are small (0.1-≤1 µm in diameter) heterogeneous vesicles released from cells constitutively or upon activation, that mediate intercellular communication. Multi-drug resistance (MDR) has been defined as the ability of cancer cells to survive after treatment with various drugs. However, the mechanism(s) used by cancer cells to evade apoptosis induced by anticancer drugs remain unclear and was the subject of our investigation. Here we report a novel mechanism of cancer cell expulsion of anticancer drugs through the release of MVs, followed by the recruitment of lysosomes to the site of release to repair the resulting damage. In addition, we show for the first time that inhibition of MV release by pretreatment of PC3M cells with the calpain inhibitor, calpeptin, sensitizes cancer cells to drug-elicited apoptosis mediated by the addition of methotrexate (MTX) and docetoxel (DOC) using at least 10-fold lower concentrations, both in vitro and in vivo. Treatment of cancer patients with MET or DOC leads to significant side effects due to the use of higher doses. Here we show that these drugs when administered together with calpeptin can be given at doses 100 times lower and still induce effective killing of target cancer cells. Overall our studies shed light on the role of MV release in cancer cell expulsion of anticancer drugs and subsequent evasion and survival from apoptosis and suggest new combination therapies for existing cancer drugs.Peer reviewedFinal Accepted Versio

Coxsackie virus entry and spread in HeLa cells is aided by microvesicle release

Microvesicles(MVs) released from plasma membrane expressing surface phosphatidylserine and ranging from 0.2-≤1 m in diameter are reported to carry various membrane proteins, lipids and cytoplasmic components characteristic of the parental cell (1). Coxsackievirus B (CVB), a member of the enterovirus family is the main cause of meningitis and encephalitis in infants which may result in neurodevelopmental defects. Calpains are calcium-dependant cysteine proteases that degrade cytoplasmic and cytoskeletal proteins. They regulate a variety of actin-dependant cellular processes such as microvesiculation. CVB1 requires calpain activation for both entry and virus replication. Here, we show that knocking down calpain, using approaches such as small interfering RNA (siRNA), culminates in reduction of MV release, as we showed before with another intracellular pathogen, the protozoan parasite, Trypanosoma cruzi (2). The reduction in MV release then abrogates CVB1 entry and spread in HeLa cells. The calpain inhibitor calpeptin also caused similar reduction in CVB1 entry and spread to healthy target cells. Together, our findings provide evidence that CVB1 infected HeLa cells enhance MV production, and these MVs aid the spread of infection. Furthermore, inhibition of MV release using siRNA results in inhibition of CVB1 entry and spread.Peer reviewedFinal Accepted Versio

The role of microvesicles in cancer and viral infection

Microvesicles are shed constitutively, or upon activation from both normal and malignant cells. Although recent studies have reported various nonlytic virus release mechanisms, this mode of virus transmission to secondary sites of infection has remained unclear. This study identified that Coxsackie virus B1 (CVB1) entry into HeLa cells results in apoptosis and production of virus-induced apoptotic microvesicles (vaMVs) by infected cells. Flow cytometery and fluorescence microscopy data illustrated that these vaMVs carry and disseminate CVB1 virions to new host cells via a non lytic MV-to-cell viral mechanism. Inhibition of MV production by siRNA knockdown of CAPNS1 in HeLa cells suggested that these vesicles mediate the spread of apoptosis to secondary sites of infection and the vaMVs could mediate non lytic MV-to-cell transmission. This thesis also identified a new mechanism for multi-drug resistance involving the efflux of anticancer drugs from cancer cells mediated by release of microvesicles, removing the drug from treated cancer cells. Immunoblotting and flow cytometery data showed that transcriptional silencing of calpain by siRNA knockdown of CAPNS1 in PC3M cells prior to drug treatment inhibits MV release and results in induced apoptosis in cells. This mechanism contributes to understanding the reasons for insensitivity to drug-induced apoptosis and the induction of drug-detoxification by cancer cells. This study has yielded important information about how to circumvent drug resistance to improve cancer chemotherapy. Furthermore, fluorescence microscopy results postulate that induction of MV release with agonist agents and anticancer drugs, results in damage to the host plasma membrane, which must be resealed immediately using activated Iysosomes if the host cell is to survive and proliferate.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Microvesiculation and Disease The role of microvesicles in cancer progression and drug resistance

Abstract Microvesicles are shed constitutively, or upon activation, from both normal and malignant cells. The process is dependent on an increase in cytosolic Ca 2 + , which activates different enzymes, resulting in depolymerization of the actin cytoskeleton and release of the vesicles. Drug resistance can be defined as the ability of cancer cells to survive exposure to a wide range of anti-cancer drugs, and anti-tumour chemotherapeutic treatments are often impaired by innate or acquired MDR (multidrug resistance). Microvesicles released upon chemotherapeutic agents prevent the drugs from reaching their targets and also mediate intercellular transport of MDR proteins

A Coxsackievirus B1-mediated nonlytic Extracellular Vesicle-to-cell mechanism of virus transmission and its possible control through modulation of EV release

Like most non-enveloped viruses, CVB1 mainly uses cell lysis to spread. Details of a nonlytic virus transmission remain unclear. Extracellular Vesicles (EVs) transfer biomolecules between cells. We show that CVB1 entry into HeLa cells results in apoptosis and release of CVB1-induced ‘medium-sized’ EVs (CVB1i-mEVs). These mEVs (100–300 nm) harbour CVB1 as shown by immunoblotting with anti-CVB1-antibody; viral capsids were detected by transmission electron microscopy and RT-PCR revealed CVB1 RNA. The percentage of mEVs released from CVB1-infected HeLa cells harbouring virus was estimated from TEM at 34 %. Inhibition of CVB1i-mEV production, with calpeptin or siRNA knockdown of CAPNS1 in HeLa cells limited spread of CVB1 suggesting these vesicles disseminate CVB1 virions to new host cells by a nonlytic EV-to-cell mechanism. This was confirmed by detecting CVB1 virions inside HeLa cells after co-culture with CVB1i-mEVs; EV release may also prevent apoptosis of infected cells whilst spreading apoptosis to secondary sites of infection