Targeting valosin‐containing protein enhances the efficacy of radiation therapy in esophageal squamous cell carcinoma

Overcoming resistance to radiation is a great challenge in cancer therapy. Here, we highlight that targeting valosin‐containing protein (VCP) improves radiation sensitivity in esophageal squamous cell carcinoma (ESCC) cell lines and show the potential of using VCP as a prognosis marker in locally advanced ESCC treated with radiation therapy. Esophageal squamous cell carcinoma cell lines with high VCP expression were treated with VCP inhibitor combined with radiotherapy. Cell proliferation, colony formation, cell death, and endoplasmic reticulum (ER) stress signaling were evaluated. Moreover, patients with newly diagnosed locally advanced ESCC who were treated with radiotherapy were analyzed. Immunohistochemistry was used to detect the expression of VCP. The correlation between overall survival and VCP was investigated. Esophageal squamous cell carcinoma cells treated with VCP inhibitor and radiotherapy showed attenuated cell proliferation and colony formation and enhanced apoptosis. Further investigation showed this combined strategy activated the ER stress signaling involved in unfolded protein response, and inhibited the ER‐associated degradation (ERAD) pathway. Clinical analysis revealed a significant survival benefit in the low VCP expression group. Targeting VCP resulted in antitumor activity and enhanced the efficacy of radiation therapy in ESCC cells in vitro. Valosin‐containing protein is a promising and novel target. In patients with locally advanced ESCC who received radiotherapy, VCP can be considered as a useful prognostic indicator of overall survival. Valosin‐containing protein inhibitors could be developed for use as effective cancer therapies, in combination with radiation therapy.     

Glutathione S‐transferase P1‐1 as a target for mesothelioma treatment

Malignant pleural mesothelioma is a poorly responsive tumor known to overexpress the phase II detoxification enzyme glutathione‐S‐transferase, which catalyzes the conjugation between glutathione and platinum(II)‐containing drugs. Therefore, we evaluated the effect of the strong glutathione S‐transferase inhibitor NBDHEX on human mesothelioma cell lines (MSTO‐211H, MPP89, MM‐B1 and Mero 48a) featuring the most common mesothelioma phenotypes: epithelioid and biphasic. Even though a different response to NBDHEX was observed, the molecule was very effective on all cell lines tested, triggering a sustained activation of both JNK and p38, followed by caspase activation and apoptosis. NBDHEX also caused severe oxidative stress in the MPP89 cells and, to a lesser extent, in the MMB1 cells, while it did not cause a significant redox imbalance in the other cell lines. The efficacy of the drug was found to be comparable or even higher than that of cisplatin. Moreover, it showed synergistic or additive effects when used in combination with cisplatin. In conclusion, NBDHEX was effective on mesothelioma cell lines, with IC₅₀ values in the low micromolar range (IC₅₀ between 1 and 4 μM). These findings indicate that NBDHEX, alone or in combination with cisplatin, is a promising new strategy for treating this rare and aggressive malignancy.     

The novel histone deacetylase inhibitor,AR‐42, inhibits gp130/Stat3 pathway and induces apoptosis and cell cycle arrest in multiple myeloma cells

Multiple myeloma (MM) remains incurable with current therapy, indicating the need for continued development of novel therapeutic agents. We evaluated the activity of a novel phenylbutyrate‐derived histone deacetylase inhibitor, AR‐42, in primary human myeloma cells and cell lines. AR‐42 was cytotoxic to MM cells at a mean LC₅₀ of 0.18 ± 0.06 μmol/l at 48 hr and induced apoptosis with cleavage of caspases 8, 9 and 3, with cell death largely prevented by caspase inhibition. AR‐42 downregulated the expression of gp130 and inhibited activation of STAT3, with minimal effects on the PI3K/Akt and MAPK pathways, indicating a predominant effect on the gp130/STAT‐3 pathway. AR‐42 also inhibited interleukin (IL)‐6‐induced STAT3 activation, which could not be overcome by exogenous IL‐6. AR‐42 also downregulated the expression of STAT3‐regulated targets, including Bcl‐xL and cyclin D1. Overexpression of Bcl‐xL by a lentivirus construct partly protected against cell death induced by AR‐42. The cyclin dependent kinase inhibitors, p16 and p21, were also significantly induced by AR‐42, which together with a decrease in cyclin D1, resulted in G₁ and G₂ cell cycle arrest. In conclusion, AR‐42 has potent cytotoxicity against MM cells mainly through gp130/STAT‐3 pathway. The results provide rationale for clinical investigation of AR‐42 in MM.     

TM‐233, a novel analog of 1′‐acetoxychavicol acetate,induces cell death in myeloma cells by inhibiting both JAK/STAT and proteasome activities

Although the introduction of bortezomib and immunomodulatory drugs has led to improved outcomes in patients with multiple myeloma, the disease remains incurable. In an effort to identify more potent and well‐tolerated agents for myeloma, we have previously reported that 1′‐acetoxychavicol acetate (ACA), a natural condiment from South‐East Asia, induces apoptotic cell death of myeloma cells in vitro and in vivo through inhibition of NF‐κB‐related functions. Searching for more potent NF‐κB inhibitors, we developed several ACA analogs based on quantitative structure–activity relationship analysis. TM‐233, one of these ACA analogs, inhibited cellular proliferation and induced cell death in various myeloma cell lines with a lower IC₅₀ than ACA. Treatment with TM‐233 inhibited constitutive activation of JAK2 and STAT3, and then downregulated the expression of anti‐apoptotic Mcl‐1 protein, but not Bcl‐2 and Bcl‐xL proteins. In addition, TM‐233 rapidly decreased the nuclear expression of NF‐κB and also decreased the accumulation of cytosolic NF‐κB. We also examined the effects of TM‐233 on bortezomib‐resistant myeloma cells that we recently established, KMS‐11/BTZ and OPM‐2/BTZ. TM‐233, but not bortezomib, inhibited cellular proliferation and induced cell death in KMS‐11/BTZ and OPM‐2/BTZ cells. Interestingly, the combination of TM‐233 and bortezomib significantly induced cell death in these bortezomib‐resistant myeloma cells through inhibition of NF‐κB activity. These results indicate that TM‐233 could overcome bortezomib resistance in myeloma cells mediated through different mechanisms, possibly inhibiting the JAK/STAT pathway. In conclusion, TM‐233 might be a more potent NF‐κB inhibitor than ACA, and could overcome bortezomib resistance in myeloma cells.     

Regulation of VCP/p97 demonstrates the critical balance between cell death and epithelial-mesenchymal transition (EMT) downstream of ER stress

Valosin-containing protein (VCP), also called p97, is a AAA+ ATPase that has been shown to be involved in endoplasmic reticulum-associated protein degradation (ERAD), mitochondria quality control and vesicle transport. We and others have previously found that disruption of VCP is sufficient to cause endoplasmic reticulum (ER) stress. We observed that induction of ER stress either following siRNA mediated loss of VCP or inhibition of VCP with eeyarestatin I potently activates an EMT-like state in cells. Interestingly, both ER stress and EMT are reversible events. Further, brief treatment of cells with eeyarestatin I increases EMT markers, and migratory and invasive properties of lung cancer cells. By examining primary lung adenocarcinoma patient samples we find that the VCP locus is heterozygously lost in nearly half of lung adenocarcinomas and VCP protein expression is decreased in nearly all primary lung tumors. Further, primary lung adenocarcinomas have increased ER stress and EMT markers. These observations have potential clinical relevance because increased ER stress and EMT markers are known to contribute to chemoresistance and poor survival of patients with lung adenocarcinoma.     

Antimyeloma activity of NK012, a micelle‐forming macromolecular prodrug of SN‐38, in an orthotopic model

NK012 is a micelle‐forming macromolecular prodrug of 7‐ethyl‐10‐hydroxy camptothecin (SN‐38), an active metabolite of irinotecan. It is accumulated and retained in tumor tissues and gradually releases SN‐38 in an enzyme‐independent manner. NK012 was previously demonstrated to have stronger antitumor activity than irinotecan in a broad range of human solid‐tumor xenograft models. In our study, we used an orthotopic multiple myeloma (MM) model created by injecting CD138‐positive U266B1, a myeloma cell line that produces human IgE lambda light chain (monoclonal protein, M protein), into immunodeficient NOD/Shi‐scid, IL‐2Rγ_c^null mice. This model shows typical bone marrow infiltration by the human myeloma cells. We evaluated the antimyeloma activity of intravenously administered NK012 in this model and showed that it suppressed the M protein concentration in the plasma and proliferation of myeloma cells in the bone marrow in a dose‐dependent manner. NK012 suppressed the progression of hind‐leg paralysis and prolonged the survival time of the mice compared to the untreated control group. In combination with bortezomib (BTZ), NK012 increased the median survival time compared to that with BTZ alone. In conclusion, these results suggest that NK012 is a potential candidate for use—alone and in combination—in the treatment of MM in humans.     

Cadmium‐coordinated supramolecule suppresses tumor growth of T‐cell leukemia in mice

Cadmium is a toxic pollutant with occupational and environmental significance, due to its diverse toxic effects. Supramolecules that conjugate and decontaminate toxic metals have potential for use in treatment of cadmium intoxication. In addition, metal‐coordinating ability has been postulated to contribute to the cytotoxic effects of anti‐tumor agents such as cisplatin or bleomycin. Thiacalixarenes, cyclic oligomers of p‐alkylphenol bridged by sulfur atoms, are supramolecules known to have potent coordinating ability to metal ions. In this study, we show that cadmium‐coordinated thiacalix[4]arene tetrasulfate (TC4ATS‐Cd) exhibits an anti‐proliferative effect against T‐cell leukemia cells. Cadmium exhibited cytotoxicity with IC₅₀ values ranging from 36 to 129 μM against epithelia‐derived cancer cell lines, while TC4ATS‐Cd elicited no significant cytotoxicity (IC₅₀ > 947 μM). However, a number of T‐cell leukemia cell lines exhibited marked sensitivity to TC4ATS‐Cd. In Jurkat cells, toxicity of TC4ATS‐Cd occurred with an IC₅₀ of 6.9 μM, which is comparable to that of 6.5 μM observed for cadmium alone. TC4ATS‐Cd induced apoptotic cell death through activation of caspase‐3 in Jurkat cells. In a xenograft model, TC4ATS‐Cd (13 mg/kg) treatment significantly suppressed the tumor growth of Jurkat cells in mice. In addition, TC4ATS‐Cd‐treated mice exhibited significantly less cadmium accumulation in liver and kidney compared to equimolar cadmium‐treated mice. These results suggest that cadmium‐coordinated supramolecules may have therapeutic potential for treatment of T‐cell leukemia.     

Anti‐proliferative and apoptosis‐inducing activity of lycopene against three subtypes of human breast cancer cell lines

Although lycopene, a major carotenoid component of tomatoes, has been suggested to attenuate the risk of breast cancer, the underlying preventive mechanism remains to be determined. Moreover, it is not known whether there are any differences in lycopene activity among different subtypes of human breast cancer cells. Using ER/PR positive MCF‐7, HER2‐positive SK‐BR‐3 and triple‐negative MDA‐MB‐468 cell lines, we investigated the cellular and molecular mechanism of the anticancer activity of lycopene. Lycopene treatment for 168 consecutive hours exhibited a time‐dependent and dose‐dependent anti‐proliferative activity against these cell lines by arresting the cell cycle at the G₀/G₁ phase at physiologically achievable concentrations found in human plasma. The greatest growth inhibition was observed in MDA‐MB‐468 where the sub‐G₀/G₁ apoptotic population was significantly increased, with demonstrable cleavage of PARP. Lycopene induced strong and sustained activation of the ERK1/2, with concomitant cyclin D1 suppression and p21 upregulation in these three cell lines. In triple negative cells, lycopene inhibited the phosphorylation of Akt and its downstream molecule mTOR, followed by subsequent upregulation of proapoptotic Bax without affecting anti‐apoptotic Bcl‐xL. Taken together, these data indicate that the predominant anticancer activity of lycopene in MDA‐MB‐468 cells suggests a potential role of lycopene for the prevention of triple negative breast cancer.     

Epstein‐Barr virus infection induces miR‐21 in terminally differentiated malignant B cells

The association of Epstein‐Barr virus (EBV) with plasmacytoid malignancies is now well established but how the virus influences microRNA expression in such cells is not known. We have used multiple myeloma (MM) cell lines to address this issue and find that an oncomiR, miR‐21 is induced after in vitro EBV infection. The PU.1 binding site in miR‐21 promoter was essential for its activation by the virus. In accordance with its noted oncogenic functions, miR‐21 induction in EBV infected MM cells caused downregulation of p21 and an increase in cyclin D3 expression. EBV infected MM cells were highly tumorigenic in SCID mice. Given the importance of miR‐21 in plasmacytoid malignancies, our findings that EBV could further exacerbate the disease by inducing miR‐21 has interesting implications both in terms of diagnosis and future miR based therapeutical approaches for the virus associated plasmacytoid tumors.     

Cannabinoid derivatives exert a potent anti‐myeloma activity both in vitro and in vivo

Although hematopoietic and immune system show high levels of the cannabinoid receptor CB2, the potential effect of cannabinoids on hematologic malignancies has been poorly determined. Here we have investigated their anti‐tumor effect in multiple myeloma (MM). We demonstrate that cannabinoids induce a selective apoptosis in MM cell lines and in primary plasma cells of MM patients, while sparing normal cells from healthy donors, including hematopoietic stem cells. This effect was mediated by caspase activation, mainly caspase‐2, and was partially prevented by a pan‐caspase inhibitor. Their pro‐apoptotic effect was correlated with an increased expression of Bax and Bak, a decrease of Bcl‐xL and Mcl‐1, a biphasic response of Akt/PKB and an increase in the levels of ceramide in MM cells. Inhibition of ceramide synthesis partially prevented apoptosis, indicating that these sphingolipids play a key role in the pro‐apoptotic effect of cannabinoids in MM cells. Remarkably, blockage of the CB2 receptor also inhibited cannabinoid‐induced apoptosis. Cannabinoid derivative WIN‐55 enhanced the anti‐myeloma activity of dexamethasone and melphalan overcoming resistance to melphalan in vitro. Finally, administration of cannabinoid WIN‐55 to plasmacytoma‐bearing mice significantly suppressed tumor growth in vivo. Together, our data suggest that cannabinoids may be considered as potential therapeutic agents in the treatment of MM.     

Tetraspanin-induced death of myeloma cell lines is autophagic and involves increased UPR signalling

Background:

Multiple myeloma (MM) therapy is hindered by the interaction of the heterogeneous malignant plasma cells with their microenvironment and evolving drug resistance. We have previously shown that the membranal tetraspanins, CD81 and CD82, are under-expressed in MM cells and that their reintroduction causes massive non-apoptotic death. In this study, we aimed to characterise the tetraspanin-induced MM death.

Methods:

Multiple myeloma cell lines were transiently transfected with eGFP–CD81N1/CD82N1 fusion proteins and assessed for death mode by flow cytometry (propidium iodide, ZVAD-fmk, 3MA), activation of unfolded protein response (UPR), and autophagy (immunoblot, RT–PCR).

Results:

Cell death induced by CD81N1 and CD82N1 in MM cell lines was autophagic and involved endoplasmic reticulum (ER)-stress manifested by activation of UPR pathways, PERK (protein kinase-like ER kinase) and IRE1 (inositol-requiring 1). We also established the relative X-box binding protein 1 baseline expression levels in a panel of MM cell lines and their general dependence on autophagy for survival. Timeline of UPR cascades and cell fate supported our results.

Interpretation:

This is the first publication implicating tetraspanins in UPR signalling pathways, autophagy, and autophagic death. Integration of our findings with published data highlights the unifying dependence of MM cells on ER–Golgi homoeostasis, and underscores the potential of tetraspanin complexes and ER-stress as leverage for MM therapy.     

Mitochondrial modulation decreases the bortezomib‐resistance in multiple myeloma cells

Multiple myeloma (MM) is an incurable hematological malignancy that causes most patients to eventually relapse and die from their disease. The 20S proteasome inhibitor bortezomib has emerged as an effective drug for MM treatment; however, intrinsic and acquired resistance to bortezomib has already been observed in MM patients. We evaluated the involvement of mitochondria in resistance to bortezomib‐induced cell death in two different MM cell lines (bortezomib‐resistant KMS20 cells and bortezomib‐sensitive KMS28BM cells). Indices of mitochondrial function, including membrane potential, oxygen consumption rate and adenosine‐5′‐triphosphate and mitochondrial Ca²⁺ concentrations, were positively correlated with drug resistance of KMS cell lines. Mitochondrial genes including CYPD, SOD2 and MCU were differentially expressed in KMS cells. Thus, changes in the expression of these genes lead to changes in mitochondrial activity and in bortezomib susceptibility or resistance, and their combined effect contributes to differential sensitivity or resistance of MM cells to bortezomib. In support of this finding, coadministration of bortezomib and 2‐methoxyestradiol, a SOD inhibitor, rendered KMS20 cells sensitive to apoptosis. Our results provide new insight into therapeutic modalities for MM patients. Studying mitochondrial activity and specific mitochondrial gene expression in fresh MM specimens might help predict resistance to proapoptotic chemotherapies and inform clinical decision‐making.     

Senescence as a main mechanism of Ritonavir and Ritonavir‐NO action against melanoma

The main focus of this study is exploring the effect and mechanism of two HIV‐protease inhibitors: Ritonavir and Ritonavir‐nitric oxide (Ritonavir‐NO) on in vitro growth of melanoma cell lines. NO modification significantly improved the antitumor potential of Ritonavir, as the IC₅₀ values of Ritonavir‐NO were approximately two times lower than IC₅₀ values of the parental compound. Our results showed for the first time, that both compounds induced senescence in primary and metastatic melanoma cell lines. This transformation was manifested as a change in cell morphology, enlargement of nuclei, increased cellular granulation, upregulation of β‐galactosidase activity, lipofuscin granules appearance, higher production of reactive oxygen species and persistent inhibition of proliferation. The expression of p53, as one of the key regulators of senescence, was upregulated after 48 hours of Ritonavir‐NO treatment only in metastatic B16F10 cells, ranking it as a late‐response event. The development of senescent phenotype was consistent with the alteration of the cytoskeleton—as we observed diminished expression of vinculin, α‐actin, and β‐tubulin. Permanent inhibition of S6 protein by Ritonavir‐NO, but not Ritonavir, could be responsible for a stronger antiproliferative potential of the NO‐modified compound. Taken together, induction of senescent phenotype may provide an excellent platform for developing therapeutic approaches based on selective killing of senescent cells.     

Protein kinase A inhibition facilitates the antitumor activity of xanthohumol,a valosin‐containing protein inhibitor

Xanthohumol (XN), a simple prenylated chalcone, can be isolated from hops and has the potential to be a cancer chemopreventive agent against several human tumor cell lines. We previously identified valosin‐containing protein (VCP) as a target of XN; VCP can also play crucial roles in cancer progression and prognosis. Therefore, we investigated the molecular mechanisms governing the contribution of VCP to the antitumor activity of XN. Several human tumor cell lines were treated with XN to investigate which human tumor cell lines are sensitive to XN. Several cell lines exhibited high sensitivity to XN both in vitro and in vivo. shRNA screening and bioinformatics analysis identified that the inhibition of the adenylate cyclase (AC) pathway synergistically facilitated apoptosis induced by VCP inhibition. These results suggest that there is crosstalk between the AC pathway and VCP function, and targeting both VCP and the AC pathway is a potential chemotherapeutic strategy for a subset of tumor cells.     

MicroRNA‐324‐5p regulates stemness,pathogenesis and sensitivity to bortezomib in multiple myeloma cells by targeting hedgehog signaling

Chromosome 17p deletions are present in 10% of patients with newly diagnosed multiple myeloma (MM), and are associated with inferior prognosis. miR‐324‐5p is located on chromosome 17p, and shows diverse functions in different types of cancers. However, its role in MM is largely unknown. Here we found the expression of miR‐324‐5p was decreased in MM, especially in del(17p) MM. In contrast, the expression of hedgehog (Hh) signaling components was elevated, indicating a correlation between miR‐324‐5p and Hh signaling in MM. Hh signaling is important for the pathogenesis of MM and maintenance of MM stem cell compartment. Indeed, overexpression of miR‐324‐5p significantly decreased Hh signaling components Smo and Gli1, and functionally reduced cell growth, survival as well as stem cell compartment in MM. Moreover, miR‐324‐5p potentiated the anti‐MM efficacy of bortezomib through regulating the activities of multidrug‐resistance proteins and the expression of Bcl‐2 family genes. Consistent results were obtained in vivo. Finally, miR‐324‐5p overcame the protective effect of bone marrow stromal cells on MM cells. Taken together, our data demonstrate that miR‐324‐5p is essential for MM pathogenesis and downregulation of miR‐324‐5p is a novel mechanism of Hh signaling activation in MM. Therefore, targeting miR‐324‐5p provides a potential therapeutic strategy for MM.     

Critical roles of T‐LAK cell‐originated protein kinase in cytokinesis

We previously reported up‐regulation of T‐LAK cell‐originated protein kinase (TOPK), a novel mitotic kinase, in the great majority of breast cancers. Here we report its critical roles in mitosis, especially in cytokinesis. We found that protein phosphatase 1 alpha (PP1α) inactivation by cyclin‐dependent kinase 1 (CDK1)/cyclin B1 caused enhancement of autophosphorylation of TOPK and resulted in its activation at an early stage of mitosis. Then TOPK interacted with and phosphorylated p97, a member of the AAA+ family of ATPase proteins, through an interaction with p47 protein as an adaptor protein. Interestingly, knockdown of TOPK or p97 in breast cancer cells caused the mitotic failures in the abscission process. This mitotic failure could be rescued by additional exogenous introduction of wild‐type TOPK protein, but not by that of its kinase‐dead form. Our findings suggest that TOPK is indispensable for cancer cell cytokinesis throughout phosphorylation on p97. (Cancer Sci 2009)