Paclitaxel induces pyroptosis by inhibiting the volume­sensitive chloride channel leucine­rich repeat­containing 8a in ovarian cancer cells.

Pyroptosis is a newly identified form of cell death, morphologically characterized by excessive cell swelling. In the present study, paclitaxel (PTX) combined with platinum were used as first­line chemotherapy, against ovarian cancer cells by inducing multiple types of cell death. However, it remains unclear whether PTX can induce pyroptosis in ovarian cancer cells. It was recently reported that PTX inhibited chloride channels, an inhibition known to cause cell swelling. In the present study, it was first verified that pyroptosis­like cell death, as well as cleaved­caspase­3 and cleaved­gasdermin E (GSDME) were induced by PTX in A2780 ovarian cancer cells. PTX inhibited the background­ and hypotonicity­activated chloride currents, promoted intracellular chloride ion accumulation, those manifestations are similar to those of the classic volume­regulatory anion channel (VRAC) blocker, 4­(2­butyl­6,7­dichloro­2­cy-clopentyl­indan­1­on5­yl) oxobutyric acid (DCPIB). Of note, both DCPIB and the downregulation of VRAC constituent protein leucine­rich repeat­containing 8a themselves could not induce persisted cell swelling and pyroptosis­like phenotypes. However, they could enhance the effects of PTX in inducing pyroptosis­like phenotypes, such as marked cell swelling, cell membrane rupture and excessive activation of caspase­3 and GSDME N­terminal fragment, which ultimately caused marked pyroptosis in A2780 cells. These findings revealed a potential mechanism of PTX and offered new insights into the effects of a synergistical combination of PTX and VRACs blockers in ovarian cancer chemotherapy.

Disulfiram-copper activates chloride currents and induces apoptosis with tyrosine kinase in prostate cancer cells.

AIM: To elucidates the mechanism that disulfiram/copper complex (DSF/Cu) treatment activates chloride channels and induces apoptosis in prostate cancer cells. METHODS: Cellular membrane currents were measured by membrane clamp technique; western blot to detect protein expression; flow cytometry to detect apoptosis; immunofluorescence to detect target protein co-localization, and further validated by a combination of protein-protein interaction and mock protein molecular docking techniques. RESULTS: DSF/Cu activated chloride channels and induced apoptosis in LNCaP (a type of androgen-dependent prostate cancer cells) cells. The chloride currents activated by DSF/Cu were significantly reduced after knockdown of CLC3 with siRNA. In addition, DSF/Cu-activated chloride currents were reduced to background current levels after perfusion with genistein, a highly specific tyrosine kinase inhibitor. Conversely, DSF/Cu failed to activate chloride currents in LNCaP cells after 30 minutes of pre-incubation with genistein. When genistein was removed, and DSF/Cu was added, the activated currents were small and unstable, and gradually decreased. Immunofluorescence in LNCaP cells also showed co-localization of the CLC3 protein with tyrosine kinase 2ß (PTK2B). CONCLUSION: DSF/Cu can activate chloride channels and induce apoptosis in LNCaP cells with the involvement of tyrosine kinase. These results provide new insights into the target therapy of prostate cancer.

ClC-3 promotes paclitaxel resistance via modulating tubulins polymerization in ovarian cancer cells.

Epithelial ovarian cancers (EOC) present as malignant tumors with high mortality in the female reproductive system diseases. Acquired resistance to paclitaxel (PTX), one of the first-line treatment of EOC, remains a therapeutic challenge. ClC-3, a member of the voltage-gated Cl- channels, plays an essential role in a variety of cellular activities, including chemotherapeutic resistance. Here, we demonstrated that the protein expression and channel function of ClC-3 was upregulated in PTX resistance A2780/PTX cells compared with its parental A2780 cells. The silence of ClC-3 expression by siRNA in A2780/PTX cells partly recovered the PTX sensitivity through restored the G2/M arrest and resumed the chloride channel blocked. ClC-3 siRNA both inhibited the expression of ClC-3 and ß-tubulin, whereas the ß-tubulin siRNA reduced the expression of itself only, without affecting the expression of ClC-3. Moreover, treatment of ClC-3 siRNA in A2780/PTX cells increased the polymerization ratio of ß-tubulin, and the possibility of proteins interaction between ClC-3 and ß-tubulin was existing. Take together, the over-expression of ClC-3 protein in PTX-resistance ovarian cancer cells promotes the combination of ClC-3 and ß-tubulin, which in turn increase the ration of free form and decrease the quota of the polymeric form of ß-tubulin, and finally reduce the sensitivity to PTX. Our findings elucidated a novel function of ClC-3 in regulating PTX resistance and ClC-3 could serve as a potential target to overcome the PTX resistance ovarian cancer.

Estrogen Receptor alpha depletion affects the biomechanical properties and cytoskeleton rearrangements in breast cancer cells.

Estrogen Receptor alpha (ERα) affects the morphology of tumors, which is closely related to the biomechanical properties and the cytoskeletal proteins. In recent years, researchers have found that biomechanical properties and cytoskeletal proteins are closely related to the occurrence and development of tumors and that biomechanical properties can be used as markers for tumor development and drug resistance. The relationship between ERα expression status and biomechanical properties, cytoskeletal proteins is not known. In this study, we found that tamoxifen-resistant breast cancer cells (MCF-7/TamR) altered cell morphology and lacked of ERα expression during the process of the Tamoxifen resistance induction. To determine whether this change was influenced by ERα expression, we transiently constructed another ERα depleted model with ERα siRNA (MCF-7/ERα siRNA) and used atomic force microscope (AFM) to detect morphological and biophysical changes. The results indicated that the roughness and Young's modulus of ERα expression depleted cells were significantly increased, accompanied by rearrangement of the cytoskeletal proteins (F-actin, FLNA, α-tubulin) and the cytoskeletal regulatory protein Rho (Rac1, CDC42) decreased. Our results have demonstrated that ERα depletion affects the biomechanical properties of breast cancer cells, which are related to cytoskeletal protein rearrangement and Rho protein decreased.

Antitumor effects of disulfiram/copper complex in the poorly-differentiated nasopharyngeal carcinoma cells via activating ClC-3 chloride channel.

The enhancement of the anticancer activity by disulfiram (DSF) chelated with copper (DSF/Cu2+) has been investigated recently, while the underlying molecular mechanisms still need to be fully elucidated. Chloride channel-3 (ClC-3) is over-expressed in a variety of cancers and involves multiple tumor biological events. However, whether the over-expression of ClC-3 in tumor cells affects the sensitivity of anti-tumor drugs remains unclear. Here, we showed that the involvement of ClC-3 chloride channel in the selective cytotoxicity of DSF/Cu2+ in the poorly-differentiated nasopharyngeal carcinoma. The EC50 of DSF alone and DSF/Cu2+ in activating the Cl- channel were 95.36 µM and 0.31 µM in the CNE-2Z cells, respectively. DSF/Cu2+ exhibited a positive correlation between the induction of the Cl- currents and the inhibition of cell proliferation. DSF/Cu2+ increased the ClC-3 protein expression and induced the cell apoptosis. Cl- channel blockers, NPPB and DIDS, and ClC-3 siRNA partially inhibited the cell apoptosis, and depleted the Cl- currents induced by DSF/Cu2+ in CNE-2Z cells. However, these effects could not be observed in the normal nasopharyngeal epithelium NP69-SV40 T cells. In vivo, the transplanted human nasopharyngeal carcinoma tumors size in the DSF/Cu2+ group decreased about 73.2% of those in the solvent control group. The chloride blockers partially inhibited the antitumor action of DSF/Cu2+. These data demonstrated that the selective cytotoxicity of DSF/Cu2+ may relate to its selective activation of ClC-3 Cl- channel pathways in CNE-2Z cells. ClC-3 Cl- channel can be viewed as a new and promising target for the treatment of nasopharyngeal carcinoma.

Different properties between spontaneous and volume-activated chloride currents in human nasopharyngeal carcinoma and its normal counterpart cells.

Although the spontaneous chloride currents (SCC) have been well studied in the normal cells, its properties and roles in neoplasms cells are still unknown. Here, we found that the SCC was manifested in the poorly differentiated human nasopharyngeal carcinoma CNE-2Z cells, with some differences such as lower occurrence and bigger current density than those of the volume-activated chloride currents (VACC). NPPB, a chloride channel blocker, inhibited the SCC much stronger than the VACC. Down-regulation of chloride channel -3 (ClC-3), a volume and mechanically dependent ion channel, could significantly decrease the VACC, but not in SCC. The occurrence, latency, and mean density of the SCC were much lower in the normal nasopharyngeal NP69-SV40T cells than those in CNE-2Z cells. Our results demonstrated that the spontaneous electrical reactivity of neoplasm cells is higher than that of normal cells, which probably relates to their high physiological activity of neoplasm cells. SIGNIFICANCE OF THE STUDY: Spontaneous chloride currents (SCC) are well known in excitable tissues and regulate a variety of physiological and pathophysiological processes. During our researching on the volume-activated chloride currents (VACC) in human nasopharyngeal carcinoma CNE-2Z cells, SCC could be also observed with different properties from VACC. Meanwhile, the occurrence, latency, and mean density of the SCC were much higher in CNE-2Z cells than those in normal nasopharyngeal NP69-SV40T cells. Our results revealed the expression and characteristics of SCC in carcinoma cells and provided a preliminary experimental basis for further exploring the function of SCC in tumour cell biology.