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Previous studies from our group have demonstrated in vitro that UCN-01 (7-hydroxystaurosporine) and inhibitors of MEK1/2 interact to cause tumor cell death in a wide variety of malignant, but not in nontransformed, cell types. The present studies determined whether UCN-01 and MEK1/2 inhibitors interacted to cause tumor cell death in vivo. In vitro colony formation studies demonstrated that UCN-01 and the MEK1/2 inhibitor PD184352 interacted to synergistically kill human mammary carcinoma cells (MDA-MB-231, MCF7) with similar combination index values. Athymic mice were implanted in the rear flank with either MDA-MB-231 or MCF7 cells and tumors permitted to form to a volume of approximately 100 mm3 prior to a two day exposure of either Vehicle, PD184352 (25 mg/kg), UCN-01 (0.1-0.2 mg/kg) or the drug combination. Tumor volume was measured every other day and tumor growth determined over the following approximately 30 days. Transient exposure of MDA-MB-231 tumors or MCF7 tumors to either PD184352 or UCN-01 did not significantly alter tumor growth rate or the mean tumor volume in vivo approximately 15-30 days after drug administration. In contrast, combined treatment with PD184352 and UCN-01 significantly reduced MDA-MB-231, and largely abolished MCF7 tumor growth. Tumor control values for both cell lines were 0.36. Tumor cells isolated approximately 30 days after combined drug exposure exhibited a significantly greater reduction in plating efficiency using ex vivo colony formation assays than tumor cells that were exposed to either drug individually. Reduced tumor growth correlated with profound tumor cell death within five days of combined drug exposure, which was also evident approximately 30 days after exposure. In addition, tumor cell death correlated with a reduction in the phosphorylation of ERK1/2 and the immuno-reactivity of Ki67 and of CD31. Collectively, these findings argue that UCN-01 and MEK1/2 inhibitors have the potential to suppress mammary tumor growth in vivo which is independent of p53 status, estrogen dependency, caspase 3 levels or oncogenic K-RAS expression.

The median survival of metastatic renal cell carcinoma (RCC) is 12 months, and the majority of treatment options are palliative. MDA-7 (interleukin-24), when expressed via a recombinant replication defective adenovirus, Ad.mda-7, has profound antiproliferative and cytotoxic effects in a wide variety of tumor cells but not in nontransformed cells. The studies in this study examined the impact of MDA-7 on RCC proliferation and survival. RCC lines (A498 and UOK121N), but not primary renal epithelial cells, were resistant to adenoviral infection that correlated with a lack of coxsackievirus and adenovirus receptor expression. Additional studies were performed using purified preparations of bacterially synthesized glutathione S-transferase (GST)-MDA-7 protein. GST-MDA-7, but not GST, caused a dose-dependent inhibition of RCC proliferation but not of primary renal epithelial cells. Clinically achievable concentrations of the novel therapeutic agent arsenic trioxide (0.5-1 micro M) were found to have little effect on RCC growth. However, the combination of GST-MDA-7 and arsenic trioxide resulted in a greater than additive reduction in cell growth that correlated with a large increase in tumor cell death. The free radical scavenger N-acetyl cysteine abolished the potentiating effect of arsenic trioxide. Although pro-caspase 3, poly(ADP-ribose) polymerase, and Bcl-(XL) levels, as well as nucleosomal DNA integrity, were reduced by combined treatment, cell killing was predominantly nonapoptotic. Combined treatment of RCC lines with GST-MDA-7 and arsenic trioxide also resulted in a substantial reduction in clonogenic survival compared with either treatment individually. Collectively, these findings demonstrate that MDA-7 protein, in combination with agents that generate free radicals, may have potential in the treatment of RCC.

Despite therapeutic interventions including surgery, chemotherapy and radiotherapy, glioblastoma multiforme (GBM) has a very poor prognosis and novel therapies are required. MDA-7 (IL-24), when expressed via a recombinant replication defective adenovirus, Ad.mda-7, has profound anti-proliferative and cytotoxic effects in a variety of tumor cells, but not in non-transformed cells. The present studies examined the combined impact of Ad.mda-7 and ionizing radiation on the proliferation and survival of GBM cells. Ad.mda-7 reduced the proliferation of rodent and human glioma cells in MTT assays and in colony formation assays. The anti-proliferative effects of Admda-7 were enhanced by radiation in a greater than additive fashion. In vitro, this cellular change correlated with enhanced cell numbers in G1/G0 and G2/M phases of the cell cycle, implying Ad.mda-7 radiosensitizes tumor cells in a cell cycle-independent manner. The radiosensitizing effects were not observed in cultures of non-transformed primary astrocytes. The enhanced reduction in growth correlated with increased necrosis and DNA degradation. Ad.mda-7 enhanced p38 and ERK1/2 activity but did not alter JNK or Akt activity. Irradiation of cells expressing MDA-7 suppressed ERK1/2 activity and dramatically enhanced JNK1/2 activity without altering either Akt or p38 activity. Inhibition of JNK1/2, but not p38, signaling abolished the radiosensitizing properties of MDA-7. Inhibition of neither ERK1/2 nor PI3K signaling enhanced the anti-proliferative effects of Ad.mda-7, whereas combined inhibition of both pathways enhanced cell killing, suggesting that ERK and PI3K signaling can be protective against MDA-7 lethality.

Prolonged activation of the mitogen-activated protein kinase (MAPK) pathway enhances expression of the cyclin kinase inhibitor p21 that can promote growth arrest and cell survival in response to cytotoxic insults. Bile acids can also cause prolonged MAPK activation that is cytoprotective against bile acid-induced cell death. Here, we examined the impact of bile acid-induced MAPK signaling and p21 expression on the survival of primary mouse hepatocytes. Deoxycholic acid (DCA) caused prolonged activation of the MAPK pathway that weakly enhanced p21 protein expression. When DCA-induced MAPK activation was blocked using MEK1/2 inhibitors, both hepatocyte viability and expression of p21 were reduced. Surprisingly, constitutive overexpression of p21 in p21+/+ hepatocytes enhanced DCA-induced cell killing. In agreement with these findings, treatment of p21-/- hepatocytes with DCA and MEK1/2 inhibitors also caused less apoptosis than observed in wild-type p21+/+ cells. Expression of p21 in p21-/- hepatocytes did not modify basal levels of apoptosis but restored the apoptotic response of p21-/- cells to those of p21+/+ cells overexpressing p21. These findings suggest that basal expression of p21 plays a facilitating, proapoptotic role in DCA-induced apoptosis. Overexpression of p21 enhanced p53 protein levels. In agreement with a role for p53 in the enhanced apoptotic response, overexpression of p21 did not potentiate apoptosis in p53-/- hepatocytes but, instead, attenuated the death response in these cells. In conclusion, our data suggest that overexpression of p21 can promote apoptosis, leading to elevated sensitivity to proapoptotic stimuli.

Recent studies have suggested that inhibition of the mitogen activated protein kinase (MAPK) pathway as well as abrogation of cell cycle check-point control can potentiate the lethal actions of chemotherapeutic drugs and radiation. We therefore investigated the impact of combined exposure to the check-point abrogator (UCN-01) in conjunction with MEK1/2 inhibitors upon survival of breast and prostate carcinoma cells. Treatment of cells with UCN-01 alone resulted in prolonged activation of the MAPK pathway. Inhibition of MEK1/2 caused modest reductions in basal MAPK activity and transiently suppressed UCN-01-stimulated MAPK activity below that of MEK1/2 inhibitor alone. Significantly, combined, but not individual, exposure of cells to UCN-01 and MEK1/2 inhibitors enhanced BAX association with mitochondria and triggered release of cytochrome c into the cytosol, accompanied by activation of effector pro-caspases, resulting in a greater than additive potentiation of apoptosis within 1 8-24h. Radiation exposure of drug treated cells did not further enhance apoptosis. Treatment of cells with both caspase 9 and caspase 8 inhibitors was required to completely inhibit apoptosis in carcinoma cells. Overexpression of Bcl-(xL) blocked cytochrome c release and cell killing induced by the drug combination. Colony forming assays demonstrated that cells exposed to both agents exhibited a substantial reduction in clonogenic survival compared to either drug alone; moreover, radiation further reduced clonogenic survival despite failing to promote additional apoptosis. Collectively, these data demonstrate that combined exposure of carcinoma cells to UCN-01 and MEK1/2 inhibitors induces apoptosis and interacts with radiation to further reduce clonogenic survival.

Recent studies have shown that inhibition of stress-induced signaling via the mitogen activated protein kinase (MAPK) pathway can potentiate the toxic effects of chemotherapeutic drugs and ionizing radiation. Because of these observations, we have further investigated the impact upon growth and survival of mammary (MDA-MB-231, MCF7, T47D), prostate (DU145, LNCaP, PC3) and squamous (A431) carcinoma cells following irradiation and combined long-term exposure to MEK1/2 inhibitors. Exposure of carcinoma cells to ionizing radiation resulted in MAPK pathway activation initially (0-4 h) and modestly enhanced MAPK activity at later times (24 h-96 h). Inhibition of radiation-induced MAPK activation using MEK1/2 inhibitors potentiated radiation-induced apoptosis in two waves, at 21-30 h and 96-144 h after exposure. The potentiation of apoptosis was not observed in MCF7, LNCaP, or PC3 cells. At 24 h, the potentiation of apoptosis was independent of radiation dose whereas at 108 h, apoptosis correlated with increasing dose. Removal of the MEK1/2 inhibitor either 6 h or 12 h after exposure abolished the potentiation of apoptosis at 24 h. At this time, the potentiation of apoptosis correlated with cleavage of pro-caspases -8, -9 and -3, and with release of cytochrome c into the cytosol. Inhibition of caspase function using a pan-caspase inhibitor ZVAD blocked the enhanced apoptotic response at 24 h. Selective inhibition of caspase 9 with LEHD or caspase 8 with IETD partially blunted the apoptotic response in MDA-MB-231, DU145 and A431 cells, whereas inhibition of both caspases reduced the response by > 90%. Removal of the MEK1/2 inhibitor either 24 h or 48 h after exposure abolished the potentiation of apoptosis at 108 h. Incubation of cells with ZVAD for 108 h also abolished the potentiation of apoptosis. In general agreement with the finding that prolonged inhibition of MEK1/2 was required to enhance radiation-induced apoptosis at 108 h, omission of MEK1/2 inhibitor from the culture media during assessment of clonogenic survival resulted in either little or no significant alteration in radiosensitivity. Collectively, our data show that combined exposure to radiation and MEK1/2 inhibitors can reduce survival in some, but not all, tumor cell types. Prolonged blunting of MAPK pathway function following radiation exposure is required for MEK1/2 inhibitors to have any effect on carcinoma cell radiosensitivity.

Interactions between the kinase inhibitor STI571 and pharmacological antagonists of the mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK)/mitogen-activated protein kinase (MAPK) cascade have been examined in human myeloid leukemia cells (K562 and LAMA 84) that express the Bcr-Abl kinase. Exposure of K562 cells to concentrations of STI571 that minimally induced apoptosis (e.g., approximately 200 nM) resulted in early suppression (i.e., at 6 h) of p42/44 MAPK phosphorylation followed at later intervals (i.e., > or =24 h) by a marked increase in p42/44 MAPK phosphorylation/activation. Coadministration of a nontoxic concentration of the MEK1/2 inhibitor PD184352 (5 microM) prevented STI571-mediated activation of p42/44 MAPK. Cells exposed to STI571 in combination with PD184352 for 48 h demonstrated a very dramatic increase in mitochondrial dysfunction (e.g., loss of DeltaPsim and cytosolic cytochrome c release) associated with procaspase-3 activation, poly(ADP-ribose) polymerase cleavage, and the appearance of the characteristic morphological features of apoptosis. Similar results were obtained using other pharmacological MEK1/2 inhibitors (e.g., PD 98059 and U0126) as well as another leukemic cell line that expresses Bcr-Abl (e.g., LAMA 84). However, synergistic induction of apoptosis by STI571 and PD184352 was not observed in human myeloid leukemia cells that do not express the Bcr-Abl kinase (e.g., HL-60 and U937) nor in normal human peripheral blood mononuclear cells. Synergistic potentiation of STI571-mediated lethality by PD184352 was associated with multiple perturbations in signaling and apoptotic regulatory pathways, including caspase-dependent down-regulation of Bcr-Abl and Bcl-2; caspase-independent down-regulation of Bcl-x(L) and Mcl-1; activation of JNK, p38 MAPK, and p34(cdc2); and diminished phosphorylation of Stat5 and CREB. Significantly, coexposure to PD184352 strikingly increased the lethality of a pharmacologically achievable concentration of STI571 (i.e., 1-2 microM) in resistant K562 cells expressing marked increases in Bcr-Abl protein levels. Together, these findings raise the possibility that treatment of Bcr-Abl-expressing cells with STI571 elicits a cytoprotective MAPK activation response and that interruption of the latter pathway (e.g., by pharmacological MEK1/2 inhibitors) is associated with a highly synergistic induction of mitochondrial damage and apoptosis. They also indicate that in the case of Bcr-Abl-positive cells, simultaneous interruption of two signal transduction pathways may represent an effective antileukemic strategy.