2,3,5,6-Tetramethylpyrazine Targets Epithelial-Mesenchymal Transition by Abrogating Manganese Superoxide Dismutase Expression and TGFß-Driven Signaling Cascades in Colon Cancer Cells.

Epithelial-mesenchymal transition (EMT) is a crucial process in which the polarized epithelial cells acquire the properties of mesenchymal cells and gain invasive properties. We have previously demonstrated that manganese superoxide dismutase (MnSOD) can regulate the EMT phenotype by modulating the intracellular reactive oxygen species. In this report, we have demonstrated the EMT-suppressive effects of 2,3,5,6-Tetramethylpyrazine (TMP, an alkaloid isolated from Chuanxiong) in colon cancer cells. TMP suppressed the expression of MnSOD, fibronectin, vimentin, MMP-9, and N-cadherin with a parallel elevation of occludin and E-cadherin in unstimulated and TGFß-stimulated cells. Functionally, TMP treatment reduced the proliferation, migration, and invasion of colon cancer cells. TMP treatment also modulated constitutive activated as well as TGFß-stimulated PI3K/Akt/mTOR, Wnt/GSK3/ß-catenin, and MAPK signaling pathways. TMP also inhibited the EMT program in the colon cancer cells-transfected with pcDNA3-MnSOD through modulation of MnSOD, EMT-related proteins, and oncogenic pathways. Overall, these data indicated that TMP may inhibit the EMT program through MnSOD-mediated abrogation of multiple signaling events in colon cancer cells.

Withanolide modulates the potential crosstalk between apoptosis and autophagy in different colorectal cancer cell lines.

Withaferin A (WFA), a withanolide, is isolated from plants of Withania somnifera (L.) Dual (Solanaceae), known as Indian ginseng, Indian winter cherry or Ashwagandha. It has been reported to exert multifaceted anti-neoplastic effects. Here, we analyzed the impact of WFA on apoptosis and autophagy activation in different human colorectal cancer cell lines. We observed that WFA exposure caused an increased aggregation of cells in the subG1 arrest in cell cycle, and increased the number of late apoptotic cells. WFA also induced the apoptosis via PARP and caspase-3 cleavage accompanied with suppression of levels of anti-apoptotic proteins like Bcl-2 and Bcl-xl. The influence of WFA on autophagy was validated by acridine orange, MDC staining, and immunocytochemistry of LC3. It was found that 24 h treatment of WFA increased the acridine and MDC stained autophagosome with induced the LC3 and other autophagy markers Atg7 and beclin-1 activation. We used Z-DEVD-FMK, a caspase-3 blocker, and 3-MA, an autophagy inhibitor, to confirm whether these effects were specific to apoptosis and autophagy, and observed the recovery of both these processes upon exposure to WFA. Moreover, the activation of ß-catenin protein was attenuated by WFA. Interestingly, small interfering RNA (siRNA)-promoted ß-catenin knockdown augmented the WFA-induced active form of p-GSK-3ß, and stimulated autophagy and apoptosis through PARP and LC3 activation. These findings suggested that WFA could stimulate activation of both apoptosis and autophagy process via modulating ß-catenin pathway.

Tanshinone IIA exerts autophagic cell death through down-regulation of ß-catenin in renal cell carcinoma cells.

Renal cell carcinoma (RCC), also called kidney cancer, is one of the most common malignancies worldwide, including the United States and China. Because of the characteristics of RCC that are both insidious and largely insensitive to chemo-radiation, the incidence and mortality of RCC are increasing every year. However, there are few studies describing anti-cancer effects of the natural compounds on RCC as compared to other cancers. Here, we analyzed the anti-neoplastic impact of Tanshinone IIA (TSN) on RCC cells. We noted that TSN increased the expression of LC3 proteins while having little effect on PARP and Alix protein expression. We found that TSN up-regulated the expression of autophagy-related proteins such as Atg7 and Beclin-1. Moreover, TSN promoted the formation of autophagic vacuoles such as autophagosomes and autolysosomes. However, treatment with 3-Methyladenine (3-MA) or Chloroquine (CQ), slightly decreased the ability of TSN to induce autophagy, but still autophagy occurred. In addition, TSN inhibited translocation of ß-catenin into the nucleus, and ß-catenin deletion and TSN treatment in RCC increased the expression of LC3 protein. Overall, our findings indicate that TSN can exert significant anti-tumor effects through down-regulation of ß-catenin to induce autophagic cell death.

Potential function of oxymatrine as a novel suppressor of epithelial-to-mesenchymal transition in lung tumor cells.

AIMS: Tumor cells metastasis as well as proliferation are important factors that can substantially determines the prognosis of cancer. In particular, epithelial-mesenchymal transition (EMT) is key phenomena which can cause tumor cell transition into other organs by promoting the disruption of the cell-cell junctions. Because oxymatrine (OMT) have been reported to attenuate the tumor growth, we investigated whether OMT can down-regulate EMT process in tumor cells. We also focused on transforming growth factor-ß (TGF-ß)-induced EMT process because EMT process can be significantly induced by this growth factor. MAIN METHODS: The cell viability was measured by MTT and real time cell analysis (RTCA) assay. The expression levels of various proteins involved in the regulation of EMT and Akt/mTOR/PI3K signaling pathway were evaluated by Western blot analysis. mRNA levels of several important EMT markers were analyzed by reverse transcription polymerase chain reaction (RT-PCR). The effects of OMT on the cellular invasion and migration were evaluated by RTCA, wound healing assay, and boyden chamber assays. KEY FINDINGS: OMT suppressed the expression of both constitutive and TGF-ß-induced mesenchymal markers, such as fibronectin, vimentin, MMP-9, MMP-2, N-cadherin, Twist, and Snail, but induced the levels of epithelial markers. Moreover, OMT down-regulated oncogenic PI3K/Akt/mTOR pathways which lead to a significant attenuation of invasive and migratory potential of lung cancer cells. SIGNIFICANCE: Overall, our study established a novel anti-metastatic role of OMT against human lung cancer cells.

Identification of Matrine as a Novel Regulator of the CXCR4 Signaling Axis in Tumor Cells.

Matrine, a quinolizidine alkaloid, is commonly employed for treating various viral and inflammatory disorders. Here, we have evaluated matrine for its activity on C-X-C chemokine receptor type 4 (CXCR4) and matrix metalloproteinases (MMP-9/2) expression, and its potential to affect tumor metastasis and invasion. The effects of matrine on CXCR4, MMP-9/2, and nuclear factor κB (NF-κB) activation in lung (A549), prostate (DU145), and pancreas (MIA PaCa-2) cells were investigated by diverse techniques. The expression level of CXCR4 and MMP-9/2 was analyzed by western blot analysis and reverse transcription polymerase chain reaction. NF-κB activation was also evaluated by western blot analysis, electrophoretic mobility shift assay as well as immunocytochemical experiments. Furthermore, we monitored cell invasion and metastasis activities by wound healing and Boyden chamber assays. We noted that matrine induced a down-regulation of CXCR4 and MMP-9/2 at both protein and mRNA levels. In addition, matrine negatively regulated human epidermal growth factor receptor 2 (HER2) and C-X-C Motif Chemokine Ligand 12 (CXCL12)-induced CXCR4 expression. Moreover, NF-κB suppression by matrine led to inhibition of metastatic potential of tumor cells. Our results suggest that matrine can block the cancer metastasis through the negative regulation of CXCR4 and MMP-9/2 and consequently it can be considered as a potential candidate for cancer therapy.

Evodiamine Mitigates Cellular Growth and Promotes Apoptosis by Targeting the c-Met Pathway in Prostate Cancer Cells.

Evodiamine (EVO) is an indoloquinazoline alkaloid that exerts its various anti-oncogenic actions by blocking phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt), mitogen-activated protein kinase (MAPK), c-Met, and nuclear factor kappa B (NF-κB) signaling pathways, thus leading to apoptosis of tumor cells. We investigated the ability of EVO to affect hepatocyte growth factor (HGF)-induced c-Met/Src/STAT3 activation cascades in castration-resistant prostate cancer (CRPC). First, we noted that EVO showed cytotoxicity and anti-proliferation activities in PC-3 and DU145 cells. Next, we found that EVO markedly inhibited HGF-induced c-Met/Src/STAT3 phosphorylation and impaired the nuclear translocation of STAT3 protein. Then, we noted that EVO arrested the cell cycle, caused apoptosis, and downregulated the expression of various carcinogenic markers such as B-cell lymphoma 2 (Bcl-2), B-cell lymphoma-extra large (Bcl-xL), cyclin D1, cyclooxygenase 2 (COX-2), survivin, vascular endothelial growth factor (VEGF), and matrix metallopeptidases 9 (MMP-9). Moreover, it was observed that in cPC-3 and DU145 cells transfected with c-Met small interfering RNA (siRNA), Src/STAT3 activation was also mitigated and led to a decrease in EVO-induced apoptotic cell death. According to our results, EVO can abrogate the activation of the c-Met/Src/STAT3 signaling axis and thus plays a role as a robust suppressor of tumor cell survival, proliferation, and angiogenesis.

Oxymatrine Attenuates Tumor Growth and Deactivates STAT5 Signaling in a Lung Cancer Xenograft Model.

Oxymatrine (OMT) is a major alkaloid found in radix Sophorae flavescentis extract and has been reported to exhibit various pharmacological activities. We elucidated the detailed molecular mechanism(s) underlying the therapeutic actions of OMT in non-small cell lung cancer (NSCLC) cells and a xenograft mouse model. Because the STAT5 signaling cascade has a significant role in regulating cell proliferation and survival in tumor cells, we hypothesized that OMT may disrupt this signaling cascade to exert its anticancer effects. We found that OMT can inhibit the constitutive activation of STAT5 by suppressing the activation of JAK1/2 and c-Src, nuclear localization, as well as STAT5 binding to DNA in A549 cells and abrogated IL-6-induced STAT5 phosphorylation in H1299 cells. We also report that a sub-optimal concentration of OMT when used in combination with a low dose of paclitaxel produced significant anti-cancer effects by inhibiting cell proliferation and causing substantial apoptosis. In a preclinical lung cancer mouse model, OMT when used in combination with paclitaxel produced a significant reduction in tumor volume. These results suggest that OMT in combination with paclitaxel can cause an attenuation of lung cancer growth both in vitro and in vivo.

Anti-myeloma Effects of Icariin Are Mediated Through the Attenuation of JAK/STAT3-Dependent Signaling Cascade.

Because of the essential role of signal transducer and activator of transcription 3 (STAT3) in proliferation, anti-apoptosis, and chemoresistance of multiple myeloma (MM), we investigated whether icariin, a prenylated flavonol glycoside, inhibits both constitutive and inducible STAT3 activation in human myeloma cell lines. We noted that icariin could block constitutive STAT3 phosphorylation as well as its nuclear translocation and DNA binding ability in U266 cells. Icariin also suppressed IL-6-induced STAT3 activation through the inhibition of upstream kinases (Janus activated kinase-1 and -2, and c-Src). We found that icariin downregulated the protein expression of STAT3 downstream target gene products such as Bcl-2, Bcl-xl, survivin, IAP-1/2, COX-2, VEGF, and matrix metallopeptidase 9 (MMP-9) in a concentration-dependent manner. Moreover, this flavonoid also exhibited the capacity to significantly induce apoptosis and suppress proliferation of MM cells. Interestingly, this agent also significantly potentiated the apoptotic effects of bortezomib through the suppression of STAT3 activation in MM cells. Altogether, our data indicates that the potential application of icariin as a STAT3 blocker in myeloma therapy.

Design, Synthesis, and Preclinical Evaluation of 4-Substituted-5-methyl-furo[2,3-d]pyrimidines as Microtubule Targeting Agents That Are Effective against Multidrug Resistant Cancer Cells.

The design, synthesis, and biological evaluations of eight 4-substituted 5-methyl-furo[2,3-d]pyrimidines are reported. Synthesis involved N(4)-alkylation of N-aryl-5-methylfuro[2,3-d]pyrimidin-4-amines, obtained from Ullmann coupling of 4-amino-5-methylfuro[2,3-d]pyrimidine and appropriate aryl iodides. Compounds 3, 4, and 9 showed potent microtubule depolymerizing activities, while compounds 6-8 had slightly lower potency. Compounds 4, 6, 7, and 9 inhibited tubulin assembly with IC50 values comparable to that of combretastatin A-4 (CA-4). Compounds 3, 4, and 6-9 circumvented Pgp and ßIII-tubulin mediated drug resistance, mechanisms that can limit the efficacy of paclitaxel, docetaxel, and the vinca alkaloids. In the NCI 60-cell line panel, compound 3 exhibited GI50 values less than 10 nM in 47 of the cell lines. In an MDA-MB-435 xenograft model, compound 3 had statistically significant antitumor effects. The biological effects of 3 identify it as a novel, potent microtubule depolymerizing agent with antitumor activity.

N2-Trimethylacetyl substituted and unsubstituted-N4-phenylsubstituted-6-(2-pyridin-2-ylethyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamines: design, cellular receptor tyrosine kinase inhibitory activities and in vivo evaluation as antiangiogenic, antimetastatic and antitumor agents.

Six novel N(4)-phenylsubstituted-6-(2-pyridin-2-ylethyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamines and their N(2)-trimethylacetyl substituted analogs were synthesized as receptor tyrosine kinase (RTK) inhibitors. A microwave-mediated Sonogashira reaction was used as a key step for the synthesis of these compounds. Biological evaluation, in whole cell assays, showed that some analogs had remarkable inhibitory activity against a variety of RTKs and in particular cytotoxic activity against A431 tumor cells in culture. The inhibitory data against RTKs in this study demonstrated that variation of the 4-anilino substituents of these analogs dictates both potency and specificity of inhibitory activity against various RTKs. The study also supported the hypothesis that interaction of substituents on the 2-amino group with hydrophobic site-II provides an increase in potency. Compound 8 of this series was selected for evaluation in vivo in a B16-F10 syngeneic mouse tumor model and exhibited significant reduction in tumor growth rate, in tumor vascular density and in metastases to the lung compared to the control.

2-Amino-4-methyl-5-phenylethyl substituted-7-N-benzyl-pyrrolo[2,3-d]pyrimidines as novel antitumor antimitotic agents that also reverse tumor resistance.

Gangjee et al. recently reported a novel series of 2-amino-4-methyl-5-phenylethyl substituted-7-benzyl-pyrrolo[2,3-d]pyrimidines, some of which exhibited two digit nanomolar antitumor and antimitotic activity and were not subject to P-glycoprotein (Pgp) or multidrug resistance protein 1 (MRP1) mediated tumor resistance (unlike the Vinca alkaloids and taxanes). Some of these compounds, in addition to their antitumor activity, had the ability to reverse the Pgp-mediated resistance to clinically used antimitotic agents. This report consists of an attempt to optimize the various activities of the parent compounds by synthetic variations of the phenyl ring of the 5-phenylethyl side chain. The target compounds were synthesized via a nine-step synthesis involving a Sonogashira reaction. The substituted phenylacetylenes as coupling partners were in turn synthesized from unactivated aryl bromides or iodides. The target compounds exhibited moderate cytotoxicity against MCF-7 tumor cells. However, most of these compounds showed improved cytotoxicity against the resistant NCI/ADR and MCF-7/VP. This study afforded an analog which reversed both Pgp-mediated as well as MRP1-mediated resistance to clinically used antimitotic agents, along with its own antimitotic mediated antitumor activity. In addition, in the NCI-60 cell line panel one of the compounds inhibited the growth of MDA-MD-435 breast cancer cell line at submicromolar concentration.

The contribution of a 2-amino group on receptor tyrosine kinase inhibition and antiangiogenic activity in 4-anilinosubstituted pyrrolo[2,3-d]pyrimidines.

Comparison between a series of pyrrolo[2,3-d]pyrimidines with and without the 2-amino group is presented in order to determine the validity of our hypothesis that inclusion of this group improves potency against receptor tyrosine kinases (RTK). The 2-amino analogs were better against epidermal growth factor receptor (EGFR) and platelet derived growth factor-beta (PDGFR-beta) in whole cell inhibition assays and in the A431 cytotoxicity assay compared to the 2-desamino analogs. However, the 2-desamino analogs were more potent inhibitors against vascular endothelial growth factor-2 (VEGFR-2) than the corresponding 2-amino compounds. In addition, none of the 2-desamino compounds exhibited better anti-angiogenic activity in the chorioallantoic membrane (CAM) assay as compared to the standard and were only micromolar inhibitors. This study validates our original hypothesis that the inclusion of a 2-amino group in pyrrolo[2,3-d]pyrimidines improves multiple RTK inhibition and antiangiogenic activity.

Design, synthesis and biological evaluation of substituted pyrrolo[2,3-d]pyrimidines as multiple receptor tyrosine kinase inhibitors and antiangiogenic agents.

Direct and indirect involvement of receptor tyrosine kinases (RTKs) in tumor growth and metastasis makes them ideal targets for anticancer therapy. A paradigm shift from inhibition of single RTK to inhibition of multiple RTKs has been recently demonstrated. We designed and synthesized eight N(4)-phenylsubstituted-6-(2-phenylethylsubstituted)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamines as homologated series of our previously published RTK inhibitors. We reasoned that increased flexibility of the side chain, which determines potency and selectivity, would improve the spectrum of RTK inhibition. These compounds were synthesized using a bis-electrophilic cyclization to afford substituted pyrrolo[2,3-d]pyrimidines followed by chlorination and substitution at the 4-position with various anilines. Five additional compounds of this series were previously reported by Gangjee et al.(1) with activities against IGFR only. Their synthesis, characterization and biological activities against a variety of other RTKs are reported in this study for the first time. The biological evaluation, in whole cell assays, showed several analogs had remarkable inhibitory activity against epithelial growth factor receptor (EGFR), vascular endothelial growth factor receptor-1 (VEGFR-1), platelet-derived growth factor receptor-beta (PDGFR-beta), the growth of A431 cells in culture, and in the chicken embryo chorioallantoic membrane (CAM) angiogenesis assay. The inhibitory data against the RTKs in this study demonstrate that variation of the 6-ethylaryl substituents as well as the N(4)-phenyl substituents of these analogs does indeed control both the potency and specificity of inhibitory activity against RTKs. In addition, homologation of the chain length of the 6-substituent from a methylene to an ethyl increases the spectrum of RTK inhibition. New multi-RTK inhibitors (8, 12) and potent inhibitors of angiogenesis (15, 19) were identified with the best compound, N(4)-(3-trifluromethylphenyl)-6-(2-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine (15), with an IC(50) value of 30nM in the CAM angiogenesis inhibition assay.