The proteasome inhibitor, bortezomib, is ineffective against many solid tumors. Nutlin-3 is a potent antagonist of human homolog of murine double minute 2/p53 interaction exhibiting promising therapeutic anti-cancer activity. In this study, we show that treatment of various p53-defective bortezomib-resistant solid tumor cells with bortezomib plus nutlin-3 induces paraptosis, which is a cell death mode accompanied by dilation of the endoplasmic reticulum (ER) and mitochondria. Bortezomib alone did not markedly alter cellular morphology, and nutlin-3 alone induced only a transient mitochondrial dilation. However, bortezomib/nutlin-3 co-treatment triggered the progressive fusion of swollen ER and the formation of megamitochondria, leading to cell death. Mechanistically, proteasomal-impairment-induced ER stress, CHOP upregulation and disruption of Ca2+ homeostasis were found to be critically involved in the bortezomib/nutlin-3-induced dilation of the ER. Our results further suggest that mitochondrial unfolded protein stress may play an important role in the mitochondrial dilation observed during bortezomib/nutlin-3-induced cell death. Collectively, these findings suggest that bortezomib/nutlin-3 perturbs proteostasis, triggering ER/mitochondria stress and irrecoverable impairments in their structure and function, ultimately leading to paraptotic cell death.

Paraptosis is a type of programmed cell death that is characterized by dilation of the endoplasmic reticulum (ER) and/or mitochondria. Since paraptosis is morphologically and biochemically different from apoptosis, understanding its regulatory mechanisms may provide a novel therapeutic strategy in malignant cancer cells that have proven resistant to conventional pro-apoptotic treatments. Relatively little is known about the molecular basis of paraptosis, but perturbations of cellular proteostasis and ion homeostasis appear to critically contribute to the process. Ca2+ transport has been shown to be important in the paraptosis induced by several natural products, metal complexes, and co-treatment with proteasome inhibitors and certain Ca2+-modulating agents. In particular, the Ca2+-mediated communication between the ER and mitochondria plays a crucial role in paraptosis. Mitochondrial Ca2+ overload from the intracellular Ca2+-flux system located at the ER–mitochondrial axis can induce mitochondrial dilation during paraptosis, while the accumulation of misfolded proteins within the ER lumen is believed to exert an osmotic force and draw water from the cytoplasm to distend the ER lumen. In this process, Ca2+ release from the ER also critically contributes to aggravating ER stress and ER dilation. This review focuses on the role of Ca2+ transport in paraptosis by summarizing the recent findings related to the actions of Ca2+-modulating paraptosis-inducing agents and discussing the potential cancer therapeutic strategies that may effectively induce paraptosis via Ca2+ signaling.

Ophiobolin A (OP-A), a fungal sesterterpene from Bipolaris oryzae, was recently shown to have anti-glioma activity. We show here that OP-A induces paraptosis-like cell death accompanied by dilation of the endoplasmic reticulum (ER) in glioma cells, and that CHOP-mediated ER stress plays a critical role in this process. OP-A-induced ER-derived dilation and cell death were found to be independent of reactive oxygen species, but were effectively blocked by various thiol antioxidants. We observed that OP-A can react with cysteinyl thiols to form Michael adducts, suggesting that the ability of OP-A to covalently modify free sulfhydryl groups on proteins may cause protein misfolding and the accumulation of misfolded proteins, leading to paraptosis-like cell death. Taken together, these results indicate that the disruption of thiol proteostasis may critically contribute to the anti-glioma activity of OP-A.

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Mitochondrial respiratory defects have been implicated in cancer progression and metastasis, but how they control tumor cell aggressiveness remains unclear. Here, we demonstrate that a mitochondrial respiratory defect induces nuclear factor-erythroid 2 like 1 (NFE2L1) expression at the transcriptional level via reactive oxygen species (ROS)-mediated STAT3 activation. We identified syntaxin 12 (STX12) as an effective downstream target of NFE2L1 by performing cDNA microarray analysis after the overexpression and depletion of NFE2L1 in hepatoma cells. Bioinformatics analysis of The Cancer Genome Atlas Liver Hepatocellular carcinoma (TCGA-LIHC) open database (n = 371) also revealed a significant positive association (r = 0.3, p = 2.49 × 10−9) between NFE2L1 and STX12 expression. We further demonstrated that STX12 is upregulated through the ROS/STAT3/NFE2L1 axis and is a key downstream effector of NFE2L1 in modulating hepatoma cell invasiveness. In addition, gene enrichment analysis of TCGA-LIHC also showed that epithelial–mesenchymal transition (EMT)-related core genes are significantly upregulated in tumors co-expressing NFE2L1 and STX12. The positive association between NFE2L1 and STX12 expression was validated by immunohistochemistry of the hepatocellular carcinoma tissue array. Finally, higher EMT gene enrichment and worse overall survival (p = 0.043) were observed in the NFE2L1 and STX12 co-expression group with mitochondrial defect, as indicated by low NDUFA9 expression. Collectively, our results indicate that NFE2L1 is a key mitochondrial retrograde signaling-mediated primary gene product enhancing hepatoma cell invasiveness via STX12 expression and promoting liver cancer progression.

The proteasome inhibitor (PI), bortezomib (Btz), is effective in treating multiple myeloma and mantle cell lymphoma, but not solid tumors. In this study, we show for the first time that lercanidipine (Ler), an antihypertensive drug, enhances the cytotoxicity of various PIs, including Btz, carfilzomib, and ixazomib, in many solid tumor cell lines by inducing paraptosis, which is accompanied by severe vacuolation derived from the endoplasmic reticulum (ER) and mitochondria. We found that Ler potentiates Btz-mediated ER stress and ER dilation, possibly due to misfolded protein accumulation, in MDA-MB 435S cells. In addition, the combination of Btz and Ler triggers mitochondrial Ca2+ overload, critically contributing to mitochondrial dilation and subsequent paraptotic events, including mitochondrial membrane potential loss and ER dilation. Taken together, our results suggest that a combined regimen of PI and Ler may effectively kill cancer cells via structural and functional perturbations of the ER and mitochondria.

Elevated Bcl-xL expression in cancer cells contributes to doxorubicin (DOX) resistance, leading to failure in chemotherapy. In addition, the clinical use of high-dose doxorubicin (DOX) in cancer therapy has been limited by issues with cardiotoxicity and hepatotoxicity. Here, we show that co-treatment with pyrrolidine dithiocarbamate (PDTC) attenuates DOX-induced apoptosis in Chang-L liver cells and human hepatocytes, but overcomes DOX resistance in Bcl-xL-overexpressing Chang-L cells and several hepatocellular carcinoma (HCC) cell lines with high Bcl-xL expression. Additionally, combined treatment with DOX and PDTC markedly retarded tumor growth in a Huh-7 HCC cell xenograft tumor model, compared to either mono-treatment. These results suggest that DOX/PDTC co-treatment may provide a safe and effective therapeutic strategy against malignant hepatoma cells with Bcl-xL-mediated apoptotic defects. We also found that induction of paraptosis, a cell death mode that is accompanied by dilation of the endoplasmic reticulum and mitochondria, is involved in this anti-cancer effect of DOX/PDTC. The intracellular glutathione levels were reduced in Bcl-xL-overexpressing Chang-L cells treated with DOX/PDTC, and DOX/PDTC-induced paraptosis was effectively blocked by pretreatment with thiol-antioxidants, but not by non-thiol antioxidants. Collectively, our results suggest that disruption of thiol homeostasis may critically contribute to DOX/PDTC-induced paraptosis in Bcl-xL-overexpressing cells.