Molecular Cancer - Latest Articles

The isoflavone metabolite 6-methoxyequol inhibits angiogenesis and suppresses tumor growth

Sofia Bellou — 2012-05-14T00:00:00Z

Background: Increased consumption of plant-based diets has been linked to the presence of certain phytochemicals, including polyphenols such as flavonoids. Several of these compounds exert their protective effect via inhibition of tumor angiogenesis. Identification of additional phytochemicals with potential antiangiogenic activity is important not only for understanding the mechanism of the preventive effect, but also for developing novel therapeutic interventions. Results: In an attempt to identify phytochemicals contributing to the well-documented preventive effect of plant-based diets on cancer incidence and mortality, we have screened a set of hitherto untested phytoestrogen metabolites concerning their anti-angiogenic effect, using endothelial cell proliferation as an end point. Here, we show that a novel phytoestrogen, 6-methoxyequol (6-ME), inhibited VEGF-induced proliferation of human umbilical vein endothelial cells (HUVE) cells, whereas VEGF-induced migration and survival of HUVE cells remained unaffected. In addition, 6-ME inhibited FGF-2-induced proliferation of bovine brain capillary endothelial (BBCE) cells. In line with its role in cell proliferation, 6-ME inhibited VEGF-induced phosphorylation of ERK1/2 MAPK, the key cascade responsible for VEGF-induced proliferation of endothelial cells. In this context, 6-ME inhibited in a dose dependent manner the phosphorylation of MEK1/2, the only known upstream activator of ERK1/2. 6-ME did not alter VEGF-induced phosphorylation of p38 MAPK or AKT, compatible with the lack of effect on VEGF-induced migration and survival of endothelial cells. Peri-tumor injection of 6-ME in A-431 xenograft tumors resulted in reduced tumor growth with suppressed neovasularization compared to vehicle controls (P < 0.01). Conclusions: 6-ME inhibits VEGF- and FGF2-induced proliferation of ECs by targeting the phosphorylation of MEK1/2 and it downstream substrate ERK1/2, both key components of the mitogenic MAPK pathway. Injection of 6-ME in mouse A-431 xenograft tumors results to tumors with decreased neovascularization and reduced tumor volume suggesting that 6-ME may be developed to a novel anti-angiogenic agent in cancer treatment.

Protective effect of the poly(ADP-ribose) polymerase inhibitor PJ34 on mitochondrial depolarization-mediated cell death in hepatocellular carcinoma cells involves attenuation of c-Jun N-terminal kinase-2 and protein kinase B/Akt activation

Balazs Radnai — 2012-05-14T00:00:00Z

Background: 2,4-Dimethoxyphenyl-E-4-arylidene-3-isochromanone (IK11) was previously described to induce apoptotic death of A431 tumor cells. In this report, we investigated the molecular action of IK11 in the HepG2 human hepatocellular carcinoma cell line to increase our knowledge of the role of poly (ADP-ribose)-polymerase (PARP), protein kinase B/Akt and mitogen activated protein kinase (MAPK) activation in the survival and death of tumor cells and to highlight the possible role of PARP-inhibitors in co-treatments with different cytotoxic agents in cancer therapy. Results: We found that sublethal concentrations of IK11 prevented proliferation, migration and entry of the cells into their G2 phase. At higher concentrations, IK11 induced reactive oxygen species (ROS) production, mitochondrial membrane depolarization, activation of c-Jun N-terminal kinase 2 (JNK2), and substantial loss of HepG2 cells. ROS production appeared marginal in mediating the cytotoxicity of IK11 since N-acetyl cysteine was unable to prevent it. However, the PARP inhibitor PJ34, although not a ROS scavenger, strongly inhibited both IK11-induced ROS production and cell death. JNK2 activation seemed to be a major mediator of the effect of IK11 since inhibition of JNK resulted in a substantial cytoprotection while inhibitors of the other kinases failed to do so. Inhibition of Akt slightly diminished the effect of IK11, while the JNK and Akt inhibitor and ROS scavenger trans-resveratrol completely protected against it. Conclusions: These results indicate significant involvement of PARP, a marginal role of ROS and a pro-apoptotic role of Akt in this system, and raise attention to a novel mechanism that should be considered when cancer therapy is augmented with PARP-inhibition, namely the cytoprotection by inhibition of JNK2.

Role of toll-like receptor 4 on the immune escape of human oral squamous cell carcinoma and resistance of cisplatin-induced apoptosis

Zujun Sun — 2012-05-14T00:00:00Z

Background: Toll-like receptor 4 (TLR4) is expressed on immune cells as a sensor that recognizes lipopolysaccharide (LPS), a microbial conserved component. It has recently been reported that the expression of TLR4 is also found in various types of tumor cells. Cisplatin is a widely used chemotherapeutic agent for oral squamous cell carcinoma (OSCC) treatment. However, the mechanisms responsible for cisplatin resistance are not well understood. Results: The present study was designed to elucidate the role of TLR4 expression in human OSCC regarding immune escape and apoptotic resistance to cisplatin. TLR4 and the myeloid differentiation primary response gene 88 (MyD88) were highly expressed in OSCC cell lines. Upon LPS stimulation both NF-kappaB and p38 MAPK pathways were activated in OSCC cell lines, followed by the production of large quantities of IL-6, IL-8 and VEGF compared with human immortalized oral epithelia cells (HIOECs). OSCC cell lines were found to be resistant to cisplatin-mediated apoptosis after pretreatment with LPS. Conclusions: Our results suggested that TLR4 was functionally expressed in human OSCC cells and development of resistance to cisplatin in human OSCC might occur through the mechanism involving TLR4 and its signaling pathway. Suppression of TLR4 and its signaling pathway might thus elevate sensitivity to cisplatin and potentially help improve the survival of patients with OSCC.

Galectin-1, a gene preferentially expressed at the tumor margin, promotes glioblastoma cell invasion

L Gerard Toussaint III — 2012-05-14T00:00:00Z

Background: High-grade gliomas, including glioblastomas (GBMs), are recalcitrant to local therapy in part because of their ability to invade the normal brain parenchyma surrounding these tumors. Animal models capable of recapitulating glioblastoma invasion may help identify mediators of this aggressive phenotype. Methods: Patient-derived glioblastoma lines have been propagated in our laboratories and orthotopically xenografted into the brains of immunocompromized mice. Invasive cells at the tumor periphery were isolated using laser capture microdissection. The mRNA expression profile of these cells was compared to expression at the tumor core, using normal mouse brain to control for host contamination. Galectin-1, a target identified by screening the resulting data, was stably over-expressed in the U87MG cell line. Sub-clones were assayed for attachment, proliferation, migration, invasion, and in vivo tumor phenotype. Results: Expression microarray data identified galectin-1 as the most potent marker (p-value 4.0 x 10-8) to identify GBM cells between tumor-brain interface as compared to the tumor core. Over-expression of galectin-1 enhanced migration and invasion in vitro. In vivo, tumors expressing high galectin-1 levels showed enhanced invasion and decreased host survival. Conclusions: In conclusion, cells at the margin of glioblastoma, in comparison to tumor core cells, have enhanced expression of mediators of invasion. Galectin-1 is likely one such mediator. Previous studies along with the current one has proven galectin-1 to be important in the migration and invasion of glioblastoma cells, in GBM neoangiogenesis and also potentially in GBM immune privilege. Targeting this molecule may offer clinical improvement to the current standard of glioblastoma therapy, i.e. radiation, temozolomide, anti-angiogenic therapy and vaccinotherapy.

Interferon-alpha enhances antitumor activities of oncolytic adenovirus-mediated IL-24 expression in hepatocellular carcinoma

Cong-Jun Wang — 2012-05-08T00:00:00Z

Background: Hepatocellular carcinoma (HCC) has a dismal 5-year-survival rate of 10%, so novel strategies are warranted. IL-24 mediates anti-tumor activity reducing STAT3 expression, which suggests that interferon (IFN) alpha may augment tumor cell lysis and reduce angiogenesis. We investigated the antitumor activity of treatment with IFN-alpha, with the oncolytic adenovirus SG600-IL-24, or the combination of both in HCC in vitro and in vivo. Results: RT-PCR, ELISA assay and Western-blot confirmed that the exogenous IL-24 gene was highly expressed in HCC cells infected with SG600-IL-24. Treatment with combined IFN-alpha and SG600-IL-24 suppressed growth and promoted apoptosis of the HepG2, MHCC97L, and HCCLM3 cell lines compared with the normal cell line L02. The combined therapy increased STAT1 and SOCS1 and apoptosis, but decreased the expression of the metastatic and angiogenic proteins MMP-2, XIAP, OPN, and VEGF, which are regulated by STAT3 in HCC cells in vitro. To assess the effects in vivo, the HCC cell line HCCLM3 was transplanted subcutaneously into the right flanks of nude mice. Mice in the IFN-alpha group, the SG600-IL-24 group, or the combined therapy group had significantly suppressed growth of the HCC xenografted tumors compared to the PBS control group of mice. Among the mice treated with the combination of IFN-alpha and SG600-IL-24, three of those eight mice had long-term survival and no evidence of a tumor. These mice also had decreased expression of the metastatic and angiogenic proteins MMP-2, XIAP, OPN, and VEGF. Conclusions: The present study demonstrated for the first time the potential antitumor activity of IFN-alpha combined with the oncolytic adenovirus SG600-IL-24 in HCC both in vitro and in vivo, and suggests its further development as a potential candidate for HCC cancer gene therapy.

Osteoblast-secreted collagen upregulates paracrine Sonic hedgehog signaling by prostate cancer cells and enhances osteoblast differentiation

Samantha Zunich — 2012-05-04T00:00:00Z

Background: Induction of osteoblast differentiation by paracrine Sonic hedgehog (Shh) signaling may be a mechanism through which Shh-expressing prostate cancer cells initiate changes in the bone microenvironment and promote metastases. A hallmark of osteoblast differentiation is the formation of matrix whose predominant protein is type 1 collagen. We investigated the formation of a collagen matrix by osteoblasts cultured with prostate cancer cells, and its effects on interactions between prostate cancer cells and osteoblasts. Results: In the presence of exogenous ascorbic acid (AA), a co-factor in collagen synthesis, mouse MC3T3 pre-osteoblasts in mixed cultures with human LNCaP prostate cancer cells or LNCaP cells modified to overexpress Shh (LNShh cells) formed collagen matrix with distinct fibril ultrastructural characteristics. AA increased the activity of alkaline phosphatase and the expression of the alkaline phosphatase gene Akp2, markers of osteoblast differentiation, in MC3T3 pre-osteoblasts cultured with LNCaP or LNShh cells. However, the AA-stimulated increase in Akp2 expression in MC3T3 pre-osteoblasts cultured with LNShh cells far exceeded the levels observed in MC3T3 cells cultured with either LNCaP cells with AA or LNShh cells without AA. Therefore, AA and Shh exert a synergistic effect on osteoblast differentiation. We determined whether the effect of AA on LNShh cell-induced osteoblast differentiation was mediated by Shh signaling. AA increased the expression of Gli1 and Ptc1, target genes of the Shh pathway, in MC3T3 pre-osteoblasts cultured with LNShh cells to at least twice their levels without AA. The ability of AA to upregulate Shh signaling and enhance alkaline phosphatase activity was blocked in MC3T3 cells that expressed a dominant negative form of the transcription factor GLI1. The AA-stimulated increase in Shh signaling and Shh-induced osteoblast differentiation was also inhibited by the specific collagen synthesis inhibitor 3,4-dehydro-L-proline. Conclusions: Matrix collagen, formed by osteoblasts in the presence of AA, potentiates Shh signaling between Shh-expressing prostate cancer cells and osteoblasts. Collagen and Shh signaling exert a synergistic effect on osteoblast differentiation, a defining event in prostate carcinoma bone metastasis. Investigations into paracrine interactions among prostate cancer cells, osteoblasts, and osteoblast-synthesized matrix proteins advance our understanding of mechanisms contributing to prostate cancer bone metastasis.

Increased tumorigenesis associated with loss of the tumor suppressor gene Cadm1

Louise van der Weyden — 2012-05-03T00:00:00Z

Background: CADM1 encodes an immunoglobulin superfamily (IGSF) cell adhesion molecule. Inactivation of CADM1, either by promoter hypermethylation or loss of heterozygosity, has been reported in a wide variety of tumor types, thus it has been postulated as a tumor suppressor gene.FindingsWe show for the first time that Cadm1 homozygous null mice die significantly faster than wildtype controls due to an increased tumor incidence (p<0.05) of predominantly lymphomas but also some solid tumors. Furthermore, tumor latency is significantly reduced after irradiation (p<0.003), suggesting there are genes that collaborate with loss of Cadm1 in tumorigenesis. To identify these co-operating genetic events, we performed a Sleeping Beauty transposon-mediated insertional mutagenesis screen in Cadm1 mice, and identified several common insertion sites (CIS) found specifically on a Cadm1-null background (not wildtype background). Conclusion: We confirm that Cadm1 is indeed a bona fide tumor suppressor gene and provide new insights into genetic partners that co-operate in Cadm1-mediated tumor suppression.

Temporally distinct roles for tumor suppressor pathways in cell cycle arrest and cellular senescence in Cyclin D1-driven tumor

Hasan Zalzali — 2012-05-01T00:00:00Z

Background: Cellular senescence represents a tumor suppressive response to a variety of aberrant and oncogenic insults. We have previously described a transgenic mouse model of Cyclin D1-driven senescence in pineal cells that opposes tumor progression. We now attempted to define the molecular mechanisms leading to p53 activation in this model, and to identify effectors of Cyclin D1-induced senescence. Results: Senescence evolved over a period of weeks, with initial hyperproliferation followed by cell cycle arrest due to ROS production leading to activation of a DNA damage response and the p53 pathway. Interestingly, cell cycle exit was associated with repression of the Cyclin-dependent kinase Cdk2. This was followed days later by formation of heterochromatin foci correlating with RB protein hypophosphorylation at Cdk4-dependent sites. In the absence of the Cdk4-inhibitor p18Ink4c, cell cycle exit was delayed but most cells eventually showed a senescent phenotype. However, tumors later arose from this premalignant, largely senescent lesion. We found that the p53 pathway was intact in tumors arising in a p18Ink4c-/- background, indicating that the two genes represent distinct tumor suppressor pathways. Upon tumor progression, both p18Ink4c-/- and p53-/- tumors showed increased Cdk2 expression. Inhibition of Cdk2 in cultured pre-tumorigenic and tumor cells of both backgrounds resulted in decreased proliferation and evidence of senescence. Conclusion: Our findings indicate that the p53 and the RB pathways play temporally distinct roles in senescence induction in Cyclin D1-expressing cells, and that Cdk2 inhibition plays a role in tumor suppression, and may be a useful therapeutic target.

Molecular mechanism implicated in Pemetrexedinduced apoptosis in human melanoma cells

Aitziber Buqué — 2012-04-26T00:00:00Z

Background: Metastatic melanoma is a lethal skin cancer and its incidence is rising every year. Itrepresents a challenge for oncologist, as the current treatment options are non-curative in themajority of cases; therefore, the effort to find and/or develop novel compounds is mandatory.Pemetrexed (Alimta(R), MTA) is a multitarget antifolate that inhibits folate-dependentenzymes: thymidylate synthase, dihydrofolate reductase and glycinamide ribonucleotideformyltransferase, required for de novo synthesis of nucleotides for DNA replication. It iscurrently used in the treatment of mesothelioma and non-small cell lung cancer (NSCLC),and has shown clinical activity in other tumors such as breast, colorectal, bladder, cervical,gastric and pancreatic cancer. However, its effect in human melanoma has not been studiedyet. Results: In the current work we studied the effect of MTA on four human melanoma cell lines A375,Hs294T, HT144 and MeWo and in two NSCLC cell lines H1299 and Calu-3. We have foundthat MTA induces DNA damage, S-phase cell cycle arrest, and caspase- dependent and -independent apoptosis. We show that an increment of the intracellular reactive oxygenspecies (ROS) and p53 is required for MTA-induced cytotoxicity by utilizing N-Acetyl-LCysteine(NAC) to blockage of ROS and p53-defective H1299 NSCLC cell line.Pretreatment of melanoma cells with NAC significantly decreased the DNA damage, p53 upregulationand cytotoxic effect of MTA. MTA was able to induce p53 expression leading toup-regulation of p53-dependent genes Mcl-1 and PIDD, followed by a postranscriptionalregulation of Mcl-1 improving apoptosis. Conclusions: We found that MTA induced DNA damage and mitochondrial-mediated apoptosis in humanmelanoma cells in vitro and that the associated apoptosis was both caspase-dependent and -independent and p53-mediated. Our data suggest that MTA may be of therapeutic relevancefor the future treatment of human malignant melanoma.

Nicotine, IFN-gamma and retinoic acid mediated induction of MUC4 in pancreatic cancer requires E2F1 and STAT-1 transcription factors and utilize different signaling cascades

Sateesh Kunigal — 2012-04-26T00:00:00Z

Background: The membrane-bound mucins are thought to play an important biological role in cell-cell andcell-matrix interactions, in cell signaling and in modulating biological properties of cancercell. MUC4, a transmembrane mucin is overexpressed in pancreatic tumors, while remainingundetectable in the normal pancreas, thus indicating a potential role in pancreatic cancerpathogenesis. The molecular mechanisms involved in the regulation of MUC4 gene are notyet fully understood. Smoking is strongly correlated with pancreatic cancer and in the presentstudy; we elucidate the molecular mechanisms by which nicotine as well as agents likeretinoic acid (RA) and interferon-gamma (IFN-gamma) induce the expression of MUC4 in pancreaticcancer cell lines CD18, CAPAN2, AsPC1 and BxPC3. Results: Chromatin immunoprecipitation assays and real-time PCR showed that transcription factorsE2F1 and STAT1 can positively regulate MUC4 expression at the transcriptional level. IFN-gammaand RA could collaborate with nicotine in elevating the expression of MUC4, utilizing E2F1and STAT1 transcription factors. Depletion of STAT1 or E2F1 abrogated the induction ofMUC4; nicotine-mediated induction of MUC4 appeared to require alpha7-nicotinic acetylcholinereceptor subunit. Further, Src and ERK family kinases also mediated the induction ofMUC4, since inhibiting these signaling molecules prevented the induction of MUC4. MUC4was also found to be necessary for the nicotine-mediated invasion of pancreatic cancer cells,suggesting that induction of MUC4 by nicotine and other agents might contribute to thegenesis and progression of pancreatic cancer. Conclusions: Our studies show that agents that can promote the growth and invasion of pancreatic cancercells induce the MUC4 gene through multiple pathways and this induction requires thetranscriptional activity of E2F1 and STAT1. Further, the Src as well as ERK signalingpathways appear to be involved in the induction of this gene. It appears that targeting thesesignaling pathways might inhibit the expression of MUC4 and prevent the proliferation andinvasion of pancreatic cancer cells.