Proteasome inhibitor PS-341 induces growth arrest and apoptosis of non-small cell lung cancer cells via the JNK/c-Jun/AP-1 signaling

Proteasome inhibitor PS-341 induces growth arrest and apoptosis of multiple myeloma (MM) cells via inactivation of NF-κB in vitro and has afforded some objective responses in individuals with relapsed, refractory MM. However, the activity of PS-341 against non-hematological malignancies remains to be fully elucidated. In this study, we found that PS-341 induced growth arrest and apoptosis of NCI-H520 and -H460 non-small cell lung cancer (NSCLC) cells in conjunction with markedly up-regulated levels of p21^waf1 and p53, and down-regulation of bcl-2 protein in these cells. Also, PS-341 caused phosphorylation of c-Jun NH₂-terminal kinase (JNK) and c-Jun, and enhanced AP-1/DNA binding activities in these cells as measured by western blotting and enzyme-linked immunosorbent assay (ELISA), respectively. Interestingly, when the JNK/ c-Jun/AP-1 signal pathway was disrupted by the JNK inhibitor SP600125, the ability of PS-341 to inhibit the growth of NSCLC cells and to up-regulate the levels of p21^waf1 in these cells was blunted, but the expression of p53 was sustained at a high level, suggesting that the JNK/c-Jun/AP-1 signal pathway might mediate the anti-lung cancer effects of PS-341, with p21^waf1 playing the central role. Thus, PS-341 might be useful for the treatment of individuals with NSCLC.     

Mechanisms of proteasome inhibitor PS-341-induced G(2)-M-phase arrest and apoptosis in human non-small cell lung cancer cell lines.

PURPOSE: PS-341 is a novel dipeptide boronic acid proteasome inhibitor with in vitro and in vivo antitumor activity that induces mechanisms of apoptosis by unknown mechanisms. EXPERIMENTAL DESIGN: Human non-small cell lung cancer cell lines were used to investigate effects PS-341 on cell proliferation, cell cycle progression, and the induction of apoptosis. RESULTS: PS-341 was 38-360-fold more cytotoxic against H460 cells when compared with the proteasome inhibitors MG-132 and PSI. Differential PS-341 cytotoxic effects were found with respect to P53 function: H322 cells (p53 mutant) were 6-fold less sensitive as compared with H460 cells (p53 wild type); and H358 cells (p53 null) were 1.6-fold more sensitive as compared with H460 cells (p53 wild type). A concentration- and time-dependent cell cycle blockade at G(2)-M phase was seen for H460 cells without any direct effects on microtubule polymerization or depolymerization. PS-341 exposure in H460 cells led to stabilization of p53, induction of p21(cip/waf-1) and MDM2 expression, an increase in cyclin B and cyclin A, and the activation of cyclin B and cyclin A kinases. MDM2 induction was found only in H460 cells, whereas in H322 and H358 cells, G(2)-M-phase arrest, p21(cip/waf-1) induction, and an increase in cyclin B1 were found. The commitment of G(2)-M-phase cells to apoptosis was verified by the activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase in drug-free medium. CONCLUSIONS: Our data suggest that the PS-341-induced G(2)-M-phase arrest may be associated with the inhibition of degradation of cell cycle regulators and that the up-regulation of p21(cip/waf-1) expression may be via p53-dependent and/or -independent pathways. The resulting disturbance of cell cycle progression leads either to growth inhibition or to the initiation of apoptotic pathways.     

The proteasome inhibitor bortezomib (PS-341) inhibits growth and induces apoptosis in primary effusion lymphoma cells

The ubiquitin-proteasome pathway is responsible for degrading many critical regulatory proteins involved in immune and inflammatory responses, control of cell growth and apoptosis. Recently, proteasome inhibitors have emerged as promising new therapeutic agents in hematological malignancies. Here we show that Bortezomib (PS-341), a proteasome-inhibitor, inhibits cellular proliferation and induces apoptosis in cell lines derived from Primary Effusion Lymphoma (PEL), a subtype of non-Hodgkin's lymphoma associated with infection by human herpes virus 8 (HHV-8). Bortezomib demonstrated more cytotoxicity against PEL cells than against cell lines derived from multiple myeloma, a disease for which is in current clinical use. Apoptosis induced by Bortezomib was associated with inhibition of the classical and alternative NF-kappaB pathways, upregulation of p53, p21 and p27 and activation of caspase cascade. Finally, treatment of PEL cells with Bortezomib exerted a synergistic or additive cytotoxic effect in combination with chemotherapeutic drugs or TRAIL. Taken together, these findings suggest that Bortezomib represents a promising agent for the treatment of PEL.     

Proteasome inhibition with PS-341 (bortezomib) in lung cancer therapy

PS-341 (bortezomib) represents a new class of therapeutics that targets the ubiquitin-proteasome pathway. It has broad-spectrum single-agent anticancer activity and can potentiate chemotherapy and radiation in preclinical models. Early phase clinical studies have shown tolerability and activity in multiple myeloma, lymphoma, prostate cancer, and lung cancers. By its mechanism of inhibiting protein degradation, PS-341 targets a wide range of pathways relevant to tumor progression and therapy resistance and can directly modulate expression of cyclins, p27^Kip1, p53, nuclear factor-κB, Bcl-2, and Bax. PS-341 is currently in phase I/II clinical development in both non-small cell lung cancer and small cell lung cancer. This article will review the preclinical and clinical experience with PS-341 as it relates to lung cancer.     

The proteasome inhibitor PS-341 inhibits growth, induces apoptosis, and overcomes drug resistance in human multiple myeloma cells

Human multiple myeloma (MM) is a presently incurable hematological malignancy, and novel biologically based therapies are urgently needed. Proteasome inhibitors represent a novel potential anticancer therapy. In this study, we demonstrate that the proteasome inhibitor PS-341 directly inhibits proliferation and induces apoptosis of human MM cell lines and freshly isolated patient MM cells; inhibits mitogen-activated protein kinase growth signaling in MM cells; induces apoptosis despite induction of p21 and p27 in both p53 wild-type and p53 mutant MM cells; overcomes drug resistance; adds to the anti-MM activity of dexamethasone; and overcomes the resistance to apoptosis in MM cells conferred by interleukin-6. PS-341 also inhibits the paracrine growth of human MM cells by decreasing their adherence to bone marrow stromal cells (BMSCs) and related nuclear factor kappaB-dependent induction of interleukin-6 secretion in BMSCs, as well as inhibiting proliferation and growth signaling of residual adherent MM cells. These data, therefore, demonstrate that PS-341 both acts directly on MM cells and alters cellular interactions and cytokine secretion in the BM millieu to inhibit tumor cell growth, induce apoptosis, and overcome drug resistance. Given the acceptable animal and human toxicity profile of PS-341, these studies provide the framework for clinical evaluation of PS-341 to improve outcome for patients with this universally fatal hematological malignancy.     

Stereotactic radiosurgery (SRS): treatment option for recurrent glioblastoma multiforme (GBM)

BACKGROUND: This article describes the results of a study of stereotactic radiosurgery (SRS) in the treatment of patients with recurrent malignant glioma. METHODS: Thirty-two patients with recurrent glioblastoma multiforme (GBM) were treated for 36 lesions with SRS from 1993 to 2001. Nineteen patients were male and 13 were female. The median age at primary diagnosis of the tumor was 56 years (range, 33-76 yrs). At the time of initial diagnosis a total neurosurgical resection was performed in 7, a subtotal resection in 21, and a biopsy in 4 patients. Histology evaluations revealed glioblastoma multiforme (WHO Grade IV) in all 32 patients. In all patients radiotherapy was performed as the first-line therapy, applied as fractionated external beam radiotherapy. The median interval between primary irradiation and reirradiation was 10 months. The median dose applied was 15 Gy (range, 10-20 Gy) prescribed to the 80% isodose line that encompassed the target volume. No concomitant chemotherapy was applied. RESULTS: Treatment was well tolerated by all patients. No acute toxicities > CTC Grade II occurred. No severe long-term toxicities including radionecrosis were observed. The median follow-up time was 13 months (range, 1-89 mo). All patients died of tumor progression during follow-up. The median overall survival from primary diagnosis of the tumor was 22 months (range, 9-133 mo). The survival rate at 1 year was 90%, and 49% and 26% at 2 and 3 years, respectively. Median overall survival after SRS was 10 months. At 6 and 12 months after SRS, survival rates were 72% and 28%, respectively. Median progression-free survival after SRS was 7 months. CONCLUSIONS: SRS offers effective treatment as a salvage therapy for a subgroup of patients with smaller lesions of recurrent GBM.     

Vitamin C inactivates the proteasome inhibitor PS-341 in human cancer cells.

PURPOSE: PS-341 (bortezomib, Velcade), the first proteasome inhibitor approved by the Food and Drug Administration for the treatment of patients with relapsed multiple myeloma, induces apoptosis in human cancer cell lines. Vitamin C (ascorbic acid) is an essential water-soluble vitamin required for many normal physiologic functions and has to be obtained through diet or supplemental tablets in humans. Here we studied the potential effect of vitamin C on the anticancer activity of PS-341 in human cancer cell lines. EXPERIMENTAL DESIGN: The effects of vitamin C on apoptosis induction by PS-341 alone and by PS-341 combined with tumor necrosis factor-related apoptosis-inducing ligand were studied. In addition, the effects of vitamin C and other antioxidants on PS-341-mediated proteasome inhibition were also examined. Finally, the direct chemical interaction between vitamin C and PS-341 was determined. RESULTS: Vitamin C abrogated the ability of PS-341 to induce apoptosis in various human cancer cell lines, to induce G(2)-M arrest, and to augment apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand. Moreover, vitamin C suppressed PS-341-mediated inhibition of proteasome activity. PS-341 itself did not induce generation of intracellular reactive oxygen species whereas other antioxidants failed to abrogate its biological activity. Importantly, we detected a direct chemical interaction between vitamin C and PS-341. CONCLUSION: Vitamin C directly binds to PS-431, thus inactivating PS-341 independent of its antioxidant activity. Our findings suggest that vitamin C may have a negative effect on PS-341-mediated anticancer activity.     

Perillyl alcohol induces apoptosis in human glioblastoma multiforme cells

Standard treatment of glioblastoma multiforme consisting of surgical resection, radiation and/or chemotherapy is rarely curative, emphasizing the need for new chemotherapeutic drugs. The monoterpene perillyl alcohol (POH) has preventive and therapeutic effects in a wide variety of pre-clinical tumor models and is currently under phase I and II clinical trials. In the present study, we analyzed its effect on human glioblastoma cell lines (U87 and A172) and a primary cell culture derived from a human glioblastoma tumor specimen (GBM-1). Using MTT, we showed that POH inhibits the viability of glioblastomas in a concentration-dependent way. Glioblastoma cell lines treated with POH showed morphological alterations characteristic of apoptosis. Analysis of cell cycle and quantification of DNA fragmentation, in cells stained with propidium iodide (PI), confirmed the apoptotic effect of POH on glioblastomas. These data support the potential usefulness of perillyl alcohol as an effective chemotherapeutic agent for patients with recurrent glioblastoma multiforme.     

Synergistic efficacy in human ovarian cancer cells by histone deacetylase inhibitor TSA and proteasome inhibitor PS-341

Histone deacetylase inhibitors and proteasome inhibitor are all emerging as new classes of anticancer agents. We chose TSA and PS-341 to identify whether they have a synergistic efficacy on human ovarian cancer cells. After incubated with 500 nM TSA or/and 40 nM PS-341, we found that combined groups resulted in a striking increase of apoptosis and G2/M blocking rates, no matter in A2780, cisplatin-sensitive ovarian cancer cell line OV2008 or its resistant variant C13*. This demonstrated that TSA interacted synergistically with PS-341, which raised the possibility that combined the two drugs may represent a novel strategy in ovarian cancer.     

PS-341 (Bortezomib) inhibits proliferation and induces apoptosis of megakaryoblastic MO7-e cells

PS-341 (Bortezomib) is a dipeptide boronic acid proteasome inhibitor with antitumor activity that induces apoptosis in different human cancer cell lines. We investigated effects of PS-341 (Bortezomib) on cell proliferation, cell cycle progression, induction of apoptosis and differentiation in a megakaryoblastic (MO7-e) cell line. PS-341 was able to retain NF-kappaB in the cytoplasm and inhibit cell growth (IC(50)=22.5 nM), in a dose/time-dependent way. This anti-proliferative activity resulted to be lineage-specific, because other leukemic cell lines (KG1a, K562/R7, HL60/DNR) were unaffected by the PS-341 treatment. Moreover, PS-341 in MO7-e induced a significant pro-apoptotic effect from 10 nM concentration (40% versus 12% in the control, p<0.05). On the other hand, at lower concentration (5 nM), Bortezomib blocked cell cycle in the G2 phase. Finally, this compound was able to down-regulate WT1 expression. No significant effects on cell differentiation were found. Because a spontaneous NF-kappaB activation has been reported in megakaryocytes from patients affected by myeloproliferative disorders, Bortezomib would so be an attractive therapeutic tool for these malignancies, including essential thrombocythemia or idiopathic myelofibrosis. Preliminary data show an inhibiting activity of Bortezomib in the megakaryocytic colonies formation. Finally, also down-regulation of the WT1 gene Bortezomib-driven could be relevant, because of the role that this gene would play in the pathogenesis of acute and chronic myeloproliferative disorders.     

Proteasome inhibitor PSI induces apoptosis in human mesothelioma cells

Malignant mesothelioma is an increasingly common tumor with an almost 100% mortality rate. It is refractory to conventional treatment. We have previously shown with SSH and microarray that the mRNA expression level of proteasome is higher in epithelioid mesothelioma cell lines than in sarcomatoid ones. This study evaluates the differential apoptotic effect of proteasome inhibitors on both of these mesothelioma sub-lines. Proteasome inhibitors show substantial anti-tumor activity in some tumor cells in vitro and in vivo, but the effects on mesothelioma cells has not been studied. The viability of mesothelioma cells was reduced in a dose- and time-dependent manner by the proteasome inhibitors tested; PSI was effective with a low dose, but higher concentrations were needed for calpain inhibitor I. The epithelioid mesothelioma cells are more sensitive to the inhibitors than the sarcomatoid ones, their IC50 after 24 h of treatment with PSI being 4 and 16 microm, respectively. Other mesothelioma cell lines show similar sensitivity. PSI seemed to decrease mesothelioma viability by inducing apoptosis, as verified by cell morphology, Western blotting analysis of caspase 3 cleavage, and flow-cytometric analysis. In conclusion, PSI, a representative agent that reduces viability and induces apoptosis of mesothelioma cells, might be useful in the treatment of patients with mesothelioma, especially of epithelioid phenotype.