Calcitriol enhances gemcitabine antitumor activity in vitro and in vivo by promoting apoptosis in a human pancreatic carcinoma model system

Gemcitabine is the standard care chemotherapeutic agent to treat pancreatic cancer. Previously we demonstrated that calcitriol (1, 25-dihydroxycholecalciferol) has significant anti-proliferative effects in vitro and in vivo in multiple tumor models and enhances the activity of a variety of chemotherapeutic agents. We therefore investigated whether calcitriol could potentiate the cytotoxic activity of gemcitabine in the human pancreatic cancer Capan-1 model system. Isobologram analysis revealed that calcitriol and gemcitabine had synergistic antiproliferative effect over a wide range of drug concentrations. Calcitriol did not reduce the CDDase activity in Capan-1 tumors nor in the livers of Capan-1 tumor bearing mice. Calcitriol and gemcitabine combination promoted apoptosis in Capan-1 cells compared with either agent alone. The combination treatment also increased the activation of caspases-8, -9, -6, and -3 in Capan-1 cells. This result was confirmed by substrate-based caspase activity assay. Akt phosphorylation was reduced by calcitriol and gemcitabine combination treatment compared to single agent treatment. However, ERK1/2 phosphorylation was not modulated by either agent alone or by the combination. Tumor regrowth delay studies showed that calcitriol in combination with gemcitabine resulted in a significant reduction of Capan-1 tumor volume compared to single agent treatment. Our study suggests that calcitriol and gemcitabine in combination promotes caspase-dependent apoptosis, which may contribute to increased anti-tumor activity compared to either agent alone.     

Calcitriol enhances gemcitabine antitumor activity in vitro and in vivo by promoting apoptosis in a human pancreatic carcinoma model system

Gemcitabine is the standard care chemotherapeutic agent to treat pancreatic cancer. Previously we demonstrated that calcitriol (1, 25-dihydroxycholecalciferol) has significant anti-proliferative effects in vitro and in vivo in multiple tumor models and enhances the activity of a variety of chemotherapeutic agents. We therefore investigated whether calcitriol could potentiate the cytotoxic activity of gemcitabine in the human pancreatic cancer Capan-1 model system. Isobologram analysis revealed that calcitriol and gemcitabine had synergistic antiproliferative effect over a wide range of drug concentrations. Calcitriol did not reduce the cytidine deaminase activity in Capan-1 tumors nor in the livers of Capan-1 tumor bearing mice. Calcitriol and gemcitabine combination promoted apoptosis in Capan-1 cells compared with either agent alone. The combination treatment also increased the activation of caspases-8, -9, -6 and -3 in Capan-1 cells. This result was confirmed by substrate-based caspase activity assay. Akt phosphorylation was reduced by calcitriol and gemcitabine combination treatment compared to single agent treatment. However, ERK1/2 phosphorylation was not modulated by either agent alone or by the combination. Tumor regrowth delay studies showed that calcitriol in combination with gemcitabine resulted in a significant reduction of Capan-1 tumor volume compared to single agent treatment. Our study suggests that calcitriol and gemcitabine in combination promotes caspase-dependent apoptosis, which may contribute to increased anti-tumor activity compared to either agent alone.Key words: calcitriol, gemcitabine, pancreatic carcinoma, apoptosis, Akt, ERK1/2     

Design and multi-step synthesis of chalcone-polyamine conjugates as potent antiproliferative agents

The aim of this study is to synthesize chalcone-polyamine conjugates in order to enhance bioavailability and selectivity of chalcone core towards cancer cells, using polyamine-based vectors. 3-hydroxy-3′,4,4′,5′-tetramethoxychalcone (1) and 3′,4,4′,5′-tetramethoxychalcone (2) were selected as parent chalcones since they were found to be efficient anti-proliferative agents on various cancer cells. A series of ten chalcone-polyamine conjugates was obtained by reacting carboxychalcones with different polyamine tails. Chalcones 1 and 2 showed a strong cytotoxic activity against two prostatic cancer (PC-3 and DU-145) and two colorectal cancer (HT-29 and HCT-116) cell lines. Then, chalcone-spermine conjugates 7d and 8d were shown to be the most active of the series and could be considered as promising compounds for colon and prostatic cancer adjuvant therapy.     

A Potent Chemotherapeutic Strategy with Eg5 Inhibitor against Gemcitabine Resistant Bladder Cancer

Development of resistance to gemcitabine is a major concern in bladder cancer therapy, and the mechanism remains unclear. Eg5 has been recently identified as an attractive target in cancer chemotherapy, so novel targeted chemotherapy with Eg5 inhibitor is expected to improve the anticancer effect in gemcitabine-resistant bladder cancer. In this research, RT112-Gr cells were 350-fold less sensitive to gemcitabine than the parental cell lines, while KU7-Gr cells were 15-fold less sensitive to gemcitabine than the parental cell lines. Human OneArray Microarray analysis was performed to obtain broad spectrum information about the genes differentially expressed in RT112 and RT112-Gr cells. The anti-proliferative activity of S(MeO)TLC, an Eg5 inhibitor, was analyzed in RT112-Gr cell lines using a cell viability assay. Furthermore, the inhibitory effect was evaluated in vivo using subcutaneous xenograft tumor model. According to the result of Human OneArray® GeneChip, RRM1 and RRM2 were up-regulated, while there was no significant change in Eg5. Trypan blue staining confirmed that in S(MeO)TLC and Gemcitabine combining S(MeO)TLC group cell viability were significantly decreased in RT112-Gr cells as compared with other groups. S(MeO)TLC and S(MeO)TLC+gemcitabine groups prominently suppressed tumor growth in comparison with other groups’ in vivo. There were no significant differences in S(MeO)TLC and gemcitabine+S(MeO)TLC group in the effect of inhibition of bladder cancer in vivo and in vitro. Our data collectively demonstrated that S(MeO)TLC represents a novel strategy for the treatment of gemcitabine resistant bladder cancer.     

Effects of the methoxy group in the side chain of debromoaplysiatoxin on its tumor-promoting and anti-proliferative activities

Debromoaplysiatoxin (DAT) is a tumor promoter isolated from sea hare and exhibits anti-proliferative activity against several cancer cell lines. To clarify key residues that are responsible for its tumor-promoting activity, we focused on the chiral methoxy group in the side chain, whose role had not yet been discussed or examined before. Demethoxy-DAT (8) was derived from DAT and we evaluated its tumor-promoting activity, anti-proliferative activity, and ability to bind to protein kinase C (PKC) isozymes. Compound 8 showed somewhat weaker tumor-promoting activity than that of DAT both in vitro and in vivo, but showed higher anti-proliferative activity against several cancer cell lines. Although the affinity to novel PKC isozymes of 8 was comparable to that of DAT, the affinity to conventional PKC isozymes decreased slightly. These results suggest that the methoxy group of DAT is one of the key residues critical for tumor-promoting activity but not for anti-proliferative activity. Since the methoxy group has little influence on the molecular hydrophobicity, this is the first report showing that structural factors other than hydrophobicity in the side chain of DAT affected its biological activities.     

Synthesis of DNA interactive C3-trans-cinnamide linked β-carboline conjugates as potential cytotoxic and DNA topoisomerase I inhibitors

A series of new C3-trans-cinnamide linked β-carboline conjugates has been synthesized by coupling between various β-carboline amines and substituted cinnamic acids. Evaluation of their anti-proliferative activity against a panel of selected human cancer cell lines such as A549 (lung cancer), MCF-7 (breast cancer), B16 (melanoma), HeLa (cervical cancer) and a normal cell line NIH3T3 (mouse embryonic fibroblast cell line), suggested that the newly designed conjugates are considerably active against all the tested cancer cell lines with IC₅₀ values 13–45?nM. Moreover, the conjugates 8v and 8x were the most active against MCF-7 cells (14.05?nM and 13.84?nM respectively) and also even potent on other cell lines tested. Further, detailed investigations such as cell cycle analysis, apoptosis induction study, topoisomerase I inhibition assay, DNA binding affinity and docking studies revealed that these new conjugates are DNA interactive topoisomerase I inhibitors.     

Development of novel benzofuran-isatin conjugates as potential antiproliferative agents with apoptosis inducing mechanism in Colon cancer

In the current work, a new set of carbohydrazide linked benzofuran-isatin conjugates (5a–e and 7a–i) was designed and synthesised. The anticancer activity for compounds (5b–d, 7a, 7b, 7d and 7g) was measured against NCI-55 human cancer cell lines. Compound 5d was the most efficient, and thus subjected to the five-dose screen where it showed excellent broad activity against almost all tested cancer subpanels. Furthermore, all conjugates (5a–e and 7a–i) showed a good anti-proliferative activity towards colorectal cancer SW-620 and HT-29 cell lines, with an excellent inhibitory effect for compounds 5a and 5d (IC₅₀ = 8.7 and 9.4 µM (5a), and 6.5 and 9.8 µM for (5d), respectively). Both compounds displayed selective cytotoxicity with good safety profile. In addition, both compounds provoked apoptosis in a dose dependent manner in SW-620 cells. Also, they significantly inhibited the anti-apoptotic Bcl2 protein expression and increased the cleaved PARP level that resulted in SW-620 cells apoptosis.     

Design and synthesis of a new generation of substituted purine hydroxamate analogs as histone deacetylase inhibitors

Histone deacetylase inhibitors have been proved to be great potential for the treatment of cancer. Recently, we designed and modified a series of substituted purine hydroxamate analogs as potent HDAC inhibitors based on our previous studies. The target compounds were investigated for their in vitro HDAC inhibitory activities and anti-proliferative activities. Results indicated that these compounds could effectively inhibit HDAC and possess obvious anti-proliferative activity against tumor cells. Promisingly, target compounds 4m and 4n outperformed SAHA in both enzymatic inhibitory activity and cellular anti-proliferative activity assay.     

New phenstatin–fatty acid conjugates: Synthesis and evaluation

New phenstatin–fatty acid conjugates have been synthesized and tested against the KB-3-1, H460, MCF-7 and HEK293 cell lines, with an increase in anti-proliferative activity being observed at the micro-molar level paralleling an increase in un-saturation in the fatty acid component.     

Effects of silk sericin on the proliferation and apoptosis of colon cancer cells

Sericin is a silk protein woven from silkworm cocoons (Bombyx mori). In animal model, sericin has been reported to have anti-tumoral action against colon cancer. The mechanisms underlying the activity of sericin against cancer cells are not fully understood. The present study investigated the effects of sericin on human colorectal cancer SW480 cells compared to normal colonic mucosal FHC cells. Since the size of the sericin protein may be important for its activity, two ranges of molecular weight were tested. Sericin was found to decrease SW480 and FHC cell viability. The small sericin had higher anti-proliferative effects than that of the large sericin in both cell types. Increased apoptosis of SW480 cells is associated with increased caspase-3 activity and decreased Bcl-2 expression. The anti-proliferative effect of sericin was accompanied by cell cycle arrest at the S phase. Thus, sericin reduced SW480 cell viability by inducing cell apoptosis via caspase-3 activation and down-regulation of Bcl-2 expression. The present study provides scientific data that support the protective effect of silk sericin against cancer cells of the colon and suggests that this protein may have significant health benefits and could potentially be developed as a dietary supplement for colon cancer prevention.     

Genetic transfer of PNAS-4 induces apoptosis and enhances sensitivity to gemcitabine in lung cancer

PNAS-4 has been demonstrated to induce apoptosis in U2OS cells. To evaluate its feasibility as a new strategy for cancer therapy, we analyzed its anti-tumor effect with or without gemcitabine in A549 lung cancer cells. MTT assay, Hoechst 33258 staining and flow cytometric analysis were used to determine the cytotoxicity of PNAS-4 alone or plus gemcitabine. The anti-tumor efficacy was further investigated in vivo with nude mice. PNAS-4 plasmid/liposome complexes were injected by tail vein every 4 days. Gemcitabine was given ip on a weekly schedule for 4 weeks. PNAS-4 alone and plus gemcitabine induced apoptosis in A549 cells in vitro. The xenograft lung cancer treated with PNAS-4 retarded growth compared with the empty vector. The combination of PNAS-4 with gemcitabine induced anti-tumor activity accompanied by an increase in apoptotic cells compared with PNAS-4 or gemcitabine alone. No other obvious toxicity was found. PNAS-4 therefore suppresses tumor growth in vivo and enhances sensitivity to gemcitabine. This suggests that the PNAS-4 gene could be a potential candidate for lung cancer therapy alone or in combination with gemcitabine.     

Identification of vibsanin A analog as a novel HSP90 inhibitor

Vibsanin A is the first natural product isolated from Viburnum awabuki and has several biological activities. We have reported that a vibsanin A analog, obtained from process of total synthesis of vibsanin A, has anti-proliferative activity against human cancer cell lines. In this study, we evaluated anti-proliferative effect of the vibsanin A analogs against various human cancer cell lines and examined molecular target of the analog in human cells. Among the vibsanin A analogs, vibsanin A analog C (VAC) showed anti-proliferative effect against various cancer cell lines, and the anti-proliferative activity was strongest among the vibsanin A analogs. Additionally, VAC fluctuated amounts of HSP90-related proteins in cells and inhibited HSP90-mediated protein refolding of luciferase in vitro. These results suggest that the anti-proliferative activity of VAC is due to HSP90 inhibition, and VAC has a potential as novel anti-cancer drug as HSP90 inhibitor.     

Synthesis of indirubin-N'-glycosides and their anti-proliferative activity against human cancer cell lines

The first indirubin-N'-glycosides were prepared based on reactions of isatin-N'-glycosides with indoxyls. The products show a significant anti-proliferative activity against various human cancer cell lines. Good results were observed for an indirubin-N'-mannoside which was shown to have medium to high anti-proliferative activity against all investigated cell lines. The highest activities and selectivities against the MCF-7 breast cancer cell line were observed for indirubin-N'-rhamnosides.     

Effects of olive oil polyphenols on fatty acid synthase gene expression and activity in human colorectal cancer cells

Oleuropein (OL) and hydroxytyrosol (HT), the main olive oil polyphenols, possess anti-proliferative effects in vitro. Fatty acid synthase, a key anabolic enzyme of biosynthesis of fatty acids, plays an important role in colon carcinoma development. Our aim was to investigate whether gene expression of FAS, as well as its enzymatic activity, is regulated by HT and OL in two human colon cancer cell lines, as HT-29 and SW620. In addition, we investigated the effects of these polyphenols on growth and apoptosis in these cells. FAS gene expression and activity in treated HT-29 and SW620 cells were evaluated by real-time PCR and radiochemical assay, respectively. Cell growth and apoptosis, after polyphenols treatment, were measured by MTT test and flow cytometry, respectively. The inhibition of proliferation, detected after HT treatment, was mediated by an inhibition of FAS expression and its enzymatic activity in SW620 cells, while the anti-proliferative effect in HT-29 cells seems to be independent from FAS. OL exerted an anti-proliferative effect only on SW620 cells with a mechanism which excluded FAS. Olive oil polyphenols used were able to induce apoptosis in both cell lines studied. The increase of apoptosis in these cells was accompanied by the block of cell cycle in the S phase. This study demonstrates that HT and OL may induce anti-proliferative and pro-apoptotic effects only in certain human colorectal cancer cell types. These effects are FAS mediated only in SW620 cells after treatment with HT.     

Zinc depletion efficiently inhibits pancreatic cancer cell growth by increasing the ratio of antiproliferative/proliferative genes

We investigated the ability of the zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) to reduce pancreatic cancer cell viability. TPEN was much more efficient to inhibit pancreatic adenocarcinoma cell growth than a panel of anti-cancer drugs, including 5-fluorouracil, irinotecan, cisplatin, edelfosine, trichostatin A, mitomycin C, and gemcitabine, the gold standard chemotherapeutic agent for pancreatic cancer. Moreover, TPEN showed a dose- and time-dependent anti-proliferative effect significantly higher on pancreatic cancer cells than on normal primary fibroblasts. This effect may be explained by a significantly higher zinc depletion by TPEN in pancreatic cancer cells as compared to fibroblasts. Cell viability reduction by TPEN was associated to both G1-phase cell cycle arrest and apoptosis, and to the increased ratio of the expression level of cyclin-Cdk inhibitor versus cyclin genes and apoptotic versus anti-apoptotic genes. Finally, we show that apoptotic cell death induced by TPEN involved mitochondrial injury and caspase 3 and caspase 8 activation. In this study, we suggest that zinc depletion may be an efficient strategy in the treatment of pancreatic cancer because of its reduced antiproliferative effect on normal cells.     

Role of fatty acid synthase in gemcitabine and radiation resistance of pancreatic cancers

Human fatty acid synthase (FASN) is a homo-dimeric protein with multi-enzymatic activity responsible for the synthesis of palmitate. FASN expression has been found to be up-regulated in multiple types of human cancers and its expression correlates with poor prognosis possibly by causing treatment resistance. In this study, we tested if FASN expression is up-regulated in human pancreatic cancers and if its higher expression level in pancreatic cancers causes intrinsic resistance to gemcitabine and radiation. We found that FASN expression is significantly up-regulated in human pancreatic cancer tissues without any correlation to age, sex, race, and tumor stage. Knocking down or over-expressing FASN significantly down- or up-regulate resistance of pancreatic cancer cell lines to both gemcitabine and radiation treatments. These findings imply that the elevated FASN expression in pancreatic cancers may contribute to unsuccessful treatments of pancreatic cancers by causing intrinsic resistance to both chemotherapy and radiation therapy.     

Study on anti-proliferative effect of benzoxathiole derivatives through inactivation of NF-κB in human cancer cells

To investigate the anti-proliferative effect of a newly discovered NF-kB inhibitor, 6,6-dimethyl-2-(phenylimino)-6,7-dihydrobenzo[d][1,3]oxathiol-4(5H)-one (1a), a series of its analogs (1b-n) were prepared and evaluated for their NF-κB inhibition and anti-proliferative activity against various human cancer cell lines. Slight variation of hydrophobicity by replacement of dimethyl group of 1a at 6-position with bulky isopropyl group and introduction of para-fluoro substitution on 2-phenyl group showed good NF-κB inhibitory activity and anti-proliferative activity. However, excessive increase in hydrophobicity with 2,4,6-trichloro substituents on phenyl group resulted in the loss of both the activities. From the SAR results, 2-phenylimino-6,7-dihydrobenzo[d][1,3]oxathiol-4(5H)-one was identified as the lead scaffold for investigating new anticancer agent through inactivation of NF-κB.     

Design,synthesis and ex-vivo release studies of colon-specific polyphosphazene–anticancer drug conjugates

Colon-specific azo based polyphosphazene–anticancer drug conjugates (11–18) have been synthesized and evaluated by ex-vivo release studies. The prepared polyphosphazene drug conjugates (11–18) are stable in acidic (pH = 1.2) buffer which showed that these polymer drug conjugates are protected from acidic environment which is the primary requirement of colon specific targeted drug delivery. The ex-vivo release profiles of polyphosphazene drug conjugates (11–18) have been performed in the presence as well as in the absence of rat cecal content. The results showed that more than 89% of parent drugs (methotrexate and gemcitabine) are released from polymeric backbone of polyphosphazene drug conjugates (14 and 18) having n-butanol (lipophilic moiety). The in-vitro cytotoxicity assay has also been performed which clearly indicated that these polymeric drug conjugates are active against human colorectal cancer cell lines (HT-29 and COLO 320 DM). The drug release kinetic study demonstrated that Higuchi’s equation is found to be best fitted equation which showed that release of drug from polymeric backbone as square root of time dependent process based on non-fickian diffusion. Therefore, the synthesized polyphosphazene azo based drug conjugates of methotrexate and gemcitabine are the potential candidates for colon targeted drug delivery system with minimal undesirable side effects.     

Selective tumor targeting by enhanced permeability and retention effect. Synthesis and antitumor activity of polyphosphazene-platinum (II) conjugates

Nanosized polyphosphazene-platinum (II) conjugates with a wide range of molecular weight from 24,000 to 115,000 were synthesized to study their tumor selectivity by enhanced permeability and retention (EPR) effect and their antitumor activity. It has been found from biodistribution study that the present polyphosphazene-Pt(II) conjugates exhibit high tumor selectivity by EPR effect with the tumor to tissue ratio (TTR) from 3.6 to 13 depending on the molecular size. These polymer conjugates have shown excellent in vivo antitumor activity against both murine and human cancer cell lines. In particular, xenograft trials of the conjugates have shown outstanding tumor inhibition effect on the stomach cancer cell line, YCC-3, which is one of the least responsive to the anticancer agents currently in clinical use, although the reason is not clearly explainable yet. The high in vivo activity seems to be attributed to the controlled-release of the antitumor active platinum (II) moiety, [GlyGluPt(dach] (dach=trans-(+/-)-1,2-diaminocyclohexane) from the phosphazene backbone by degradation in aqueous solution.     

Novologues containing a benzamide side chain manifest anti-proliferative activity against two breast cancer cell lines

Hsp90 represents a promising target for the development of both anti-cancer and neuroprotective agents. Structure–activity relationship studies on novobiocin and novobiocin analogues, led to the development of KU-32 and recently, KU-596, as lead compounds for the potential treatment of neurodegenerative diseases. Similar to KU-32, we have demonstrated that upon replacement of the acetamide side chain present in KU-32 with a benzamide, this neuroprotective agent was transformed into a scaffold that manifests anti-proliferative activity. To assess structure–activity relationships for this new scaffold, a library of benzamide-containing novologues was prepared and evaluated against two breast cancer cell lines. Compound 14a manifested the most potent anti-proliferative activity from these studies and induced Hsp90-dependent client protein degradation in a concentration-dependent manner.