Disrupted Mitochondrial Homeostasis Mediates The Antitumor Effects Of Microtubule-targeting Agents

Reactive oxygen species(ROS)are closely associated with cancer initiation and progression.Cancer cells usually rely on a higher level of ROS to sustain their malignant state.However,excess ROS may cause oxidative stress,resulting in cell senescence,cell death and genomic instability.Many anti-cancer treatments exert their therapeutic effect by increasing ROS levels in cancer cells.Previous study showed that oxidative stress induced by some anticancer treatments may depend on cell cycle arrest.Mitochondria are the main source of ROS and the "energy factories" of cells.Mitochondria undergo biogenesis,fusion,fission and mitophagy to maintain mitochondrial homeostasis and respond to changes in cellular function.Disruption of mitochondrial homeostasis leads to oxidative stress,energy metabolism dysfunction and is closely associated with many diseases.However,the interrelationships between cell cycle progression,mitochondrial dynamics and oxidative stress remain unclear.In this study,the microtubule-targeting drugs TH287 and paclitaxel were used to explore the relationships between M arrest,oxidative stress and mitochondrial homeostasis.TH287 or paclitaxel was found to induce a striking M arrest in HT1080(fibrosarcoma)and U2OS(osteosarcoma)cells.Meanwhile,TH287 or paclitaxel could increase the levels of the total ROS and mitochondrial ROS.Transmission electron microscopy revealed that there were more mitochondria in TH287-treated cells than in control cells,and vacuolation was apparent in some mitochondria.TH287 treatment also caused a loss of mitochondrial membrane potential(MMP).Moreover,there was an increased level of SUMO-conjugated proteins and the global acetylation,and a higher glycolytic capacity in TH287-treated cells.Together,these data indicate that mitochondria that accumulate in TH287-treated cells are functionally compromised.UCN-01,a CHK1 inhibitor,largely relieved M arrest,reduced the mitochondrial accumulation,and lowered the ROS level caused by TH287 or paclitaxel.UCN-01 was found to partially restore the loss of MMP,reduce the level of SUMO-conjugated proteins,the global acetylation and the glycolytic capacity.Similar results were obtained with cells subjected to CHK1 RNAi.The data shown above indicate that CHK1 functional disruption could spare the M arrest and attenuate mitochondrial accumulation and the associated oxidative stress.Therefore,impairment of mitochondrial homeostasis may contribute to increased oxidative stress.Then,pre-depletion of mitochondria and depletion of PGC-1α by RNAi were employed to explore the effects of anti-microtubule agents on mitochondrial accumulation and ROS.Carbonyl cyanide 3-chlorophenylhydrazone(CCCP)is a oxidative phosphorylation(OXPHOS)uncoupler,which causes the dissipation of MMP and consequently mitophagy.However,the CCCP treatment abolished the induction of M arrest and led to decreased levels of ROS.Similarly,the pre-treatment with CCCP precluded the accumulation of mitochondria and mitochondrial ROS caused by paclitaxel.When cells were depleted of Peroxisome proliferator-activated receptor-gamma co-activator-1 alpha(PGC-1α)by RNAi,their cell cycle progression was also decelerated,resulting in fewer cells arrested at M phase in response to TH287 or paclitaxel.These results further suggest that blockade of mitochondrial supply can decelerate cell cycle,and evade M arrest-coupled disruption of mitochondrial homeostasis and oxidative stress.Further studies showed that M arrest occurs prior to the emergence of mitochondrial accumulation and the associated increase of mitochondrial ROS,and UCN-01 treatment is not effective when being added after the mitochondrial accumulation and oxidative stress had occurred.Treatment with UCN-01,the depletion of CHK1,pre-depletion of mitochondria,or blocking mitochondrial biogenesis by PGC-1α depletion could each decelerate cell cycle progression.Additionally,UCN-01 could significantly alleviate oxidative DNA damage and increase the survival and proliferation of cancer cells.Antioxidant N-acetylcysteine(NAC)and Mitoquinone(MitoQ)significantly attenuated oxidative DNA damage and partially relieved the impaired proliferation of TH287-treated cancer cells.More importantly,pre-treatment with CCCP and the depletion of PGC-1α by RNAi could also reduce the level of oxidative DNA damage.Together,these results suggest that TH287 or paclitaxel induces M arrest-coupled dysfunctional mitochondrial accumulation and oxidative stress,which ultimately leads to oxidative DNA damage that affects cell survival and proliferation.When cell cycle progression is decelerated by the treatment with UCN-01,pre-depletion of mitochondria,or the knockdown of PGC-1α,cancer cells may protect the cells from the cytotoxic effect of microtubule-targeting drugs.This thesis next examined whether the antitumor effects of TH287 in vivo depend on M arrest-coupled mitochondrial accumulation.TH287 can greatly inhibit the growth of tumor xenografts,while its tumor-suppressive effect was significantly attenuated by UCN-01 when used in combination.The experimental results showed that the level of yH2AX and mitochondrial components NDUFB8 and Cytochrome C were significantly increased by TH287 treatment,but remained low when treated with TH287 and UCN-01 in combination.In summary,this study revealed that both TH287 and paclitaxel induced M arrest-coupled mitochondrial accumulation and oxidative stress,and consequently exerted genotoxic effect and impaired cell proliferation.Although the mitochondrial mass was greatly increased in cells arrested at M phase,the mitochondria were functionally compromised.When cell cycle is decelerated,via the treatment with UCN-01,the depletion of CHK1,pre-depletion of mitochondria,or blocking mitochondrial biogenesis by PGC-1αdepletion,the cells were spared from M arrest and the associated mitochondrial oxidative stress and oxidative DNA damage,and were thus more resistant to the microtubule-targeting drugs.Together,these results suggest a causal relationship between M arrest and the disruption of mitochondrial homeostasis,and that the exacerbation of oxidative stress by the dysfunctional mitochondria serves as a critical mediator of some cancer therapeutic agents.