TIMP3 Gene Polymorphisms of -1296 T > C and -915 A > G Increase the Susceptibility to Arsenic-Induced Skin Cancer: A Cohort Study and In Silico Analysis of Mutation Impacts

Long-term exposure to arsenic may induce several human cancers, including non-melanoma skin cancer. The tissue inhibitor of metalloproteinase (TIMP)-3, encoded by the TIMP3 gene, may inhibit tumor growth, invasion, and metastasis of several cancer types. In this study, we aimed to investigate effects of the TIMP3 -1296 T > C (rs9619311) and -915 A > G (rs2234921) single-nucleotide polymorphisms (SNPs) on skin cancer risk in an arsenic-exposed population, and to evaluate the influence of allele-specific changes by an in silico analysis. In total, 1078 study participants were followed up for a median of 15 years for newly diagnosed skin cancer. New cases were identified through linkage to the National Cancer Registry of Taiwan. A Cox regression analysis was used to evaluate the effects of TIMP3 variants. Transcription factor (TF) profiling of binding sites of allele-specific changes in SNPs was conducted using the JASPAR scan tool. We observed borderline associations between TIMP3 genotypes and skin cancer risk. However, when combined with high arsenic exposure levels, the rs9619311 C allele, rs2234921 G allele, or C-G haplotype groups exhibited a greater risk of developing skin cancer compared to the respective common homozygous genotype group. The in silico analysis revealed several TF motifs located at or flanking the two SNP sites. We validated that the C allele of rs9619311 attenuated the binding affinity of BACH2, MEIS2, NFE2L2, and PBX2 to the TIMP3 promoter, and that the G allele of rs2234921 reduced the affinity of E2F8 and RUNX1 to bind to the promoter. Our findings suggest significant modifications of the effect of the association between arsenic exposure and skin cancer risk by the TIMP3 rs9619311 and rs2234921 variants. The predicted TFs and their differential binding affinities to the TIMP3 promoter provide insights into how TIMP3 interacts with arsenic through TFs in skin cancer formation.

Interleukin-1 Receptor Type 2 Acts with c-Fos to Enhance the Expression of Interleukin-6 and Vascular Endothelial Growth Factor A in Colon Cancer Cells and Induce Angiogenesis.

Interleukin-1 receptor type 2 (IL1R2) acts as a decoy receptor of exogenous IL-1; however, its intracellular activity is poorly understood. We previously demonstrated that IL1R2 intracellularly activates the expression of several proinflammatory cytokines and affects cell migration. In this study, we found that intracellular IL1R2 expression was increased in human colorectal cancer cells (CRCs) compared with normal colon cells. We also observed that the mRNA levels of IL1R2 were highly correlated with IL-6 in tumor tissues of CRC patients. By modulating its expression in CRC cells, we verified that enhanced IL1R2 expression transcriptionally activated the expression of IL-6 and VEGF-A. Conditioned medium harvested from IL1R2-overexpressing CRC cells contained higher levels of IL-6 and VEGF-A than that from vector control cells and significantly enhanced the proliferation, migration, and tube formation of cultured endothelial cells. We further demonstrated a positive association of intracellular IL1R2 levels with tumor growth and microvessel density in xenograft mouse models. These results revealed that IL1R2 activates the expression of angiogenic factors. Mechanistically, we revealed that IL1R2 complexes with c-Fos and binds to the AP-1 site at the IL-6 and VEGF-A promoters. Together, these results reveal a novel function of intracellular IL1R2 that acts with c-Fos to enhance the transcription of IL-6 and VEGF-A, which promotes angiogenesis in CRC.

Effect of heme oxygenase-1 gene promoter polymorphism on cancer risk by histological subtype: A prospective study in arseniasis-endemic areas in Taiwan.

Heme oxygenase (HO)-1 is upregulated by many stressful stimuli, including arsenic. A GT-repeat ((GT)n) polymorphism in the HO-1 gene promoter inversely modulates the levels of HO-1 induction. Previous HO-1 (GT)n polymorphism studies in relation to cancer risk have shown disparate results. We prospectively investigated the associations between HO-1 (GT)n polymorphism and cancer risk related to arsenic from drinking water. Totally, 1,013 participants from community-based cohorts of arseniasis-endemic areas in Taiwan were followed for 13 years. Allelic polymorphisms were classified into long (L, ≥ 27 (GT)n) and short (S, <27 (GT)n). Newly developed cases were identified through linkage with National Cancer Registry of Taiwan. Multivariate Cox proportional hazard methods were used to evaluate effects of the HO-1 polymorphism alone or combined with arsenic exposure. Results showed that participants with the S/S genotype had an increased risk of Bowen's disease (HR = 10.49; 95% CI: 2.77-39.7), invasive skin cancer (HR = 2.99; 95% CI: 1.13-7.87), and lung squamous cell carcinoma (HR = 3.39; 95% CI: 1.15-9.95) versus those with L/S or L/L genotype. The S/S genotype combined with high arsenic exposure (>300 µg/L) had a greater risk of skin cancer compared to the genotype alone. Consistent with previous findings, participants with the S-allele had a reduced risk of lung adenocarcinoma (HR = 0.21; 95% CI: 0.03-0.68) versus those with L/L genotype. There were no significant differences in risk of urothelial carcinoma among the three genotypes. Associations of HO-1 (GT)n polymorphism with cancer risk differs by histological subtype and the polymorphism should be considered a modifier in the risk assessment of arsenic exposure.

Tracking and Finding Slow-Proliferating/Quiescent Cancer Stem Cells with Fluorescent Nanodiamonds.

Quiescent cancer stem cells (CSCs) have long been considered to be a source of tumor initiation. However, identification and isolation of these cells have been hampered by the fact that commonly used fluorescent markers are not sufficiently stable, both chemically and photophysically, to allow tracking over an extended period of time. Here, it is shown that fluorescent nanodiamonds (FNDs) are well suited for this application. Genotoxicity tests of FNDs with comet and micronucleus assays for human fibroblasts and breast cancer cells indicate that the nanoparticles neither cause DNA damage nor impair cell growth. Using AS-B145-1R breast cancer cells as the model cell line for CSC, it is found that the FND labeling outperforms 5-ethynyl-2'-deoxyuridine (EdU) and carboxyfluorescein diacetate succinimidyl ester (CFSE) in regards to its long-term tracking capability (>20 d). Moreover, through a quantification of their stem cell activity by measuring mammosphere-forming efficiencies (MFEs) and self-renewal rates, the FND-positive cells are identified to have an MFE twice as high as that of the FND-negative cells isolated from the same dissociated mammospheres. Thus, the nanoparticle-based labeling technique provides an effective new tool for tracking and finding slow-proliferating/quiescent CSCs in cancer research.

Increased susceptibility of H-Ras(G12V)-transformed human urothelial cells to the genotoxic effects of sodium arsenite.

Inorganic arsenite (iAs) is a human carcinogen. Numerous studies have shown that mutation-activated H-Ras is frequently observed in human urothelial carcinomas. The interaction between iAs, an environmental factor, and H-Ras, an oncogene, is not clear. In this study, we explored the genotoxic effects of iAs in human urothelial cells ectopically expressing H-Ras (G12V) an activated H-Ras oncogene. Our results showed that H-Ras(G12V)-transformed human urothelial cells (HUC-RAS) were more susceptible to arsenite-induced cell death, DNA damage, micronuclei formation and anchorage-independent growth than control cells (HUC-neo). Furthermore, iAs treatment induced higher intracellular levels of reactive oxygen species (ROS) in the HUC-RAS cells than in the HUC-neo cells. N-acetyl-L-cysteine could suppress the iAs-induced increases in ROS and genetic damage. We further demonstrated that the intracellular glutathione levels were significantly elevated by the iAs treatment of the HUC-neo cells, but that this effect was not observed in the HUC-RAS cells. The iAs treatment induced higher superoxide dismutase activity in the HUC-neo cells than in the HUC-RAS cells. Alternatively, catalase activity was higher in the HUC-RAS cells than in the HUC-neo cells, but this enzyme was significantly suppressed by iAs. Moreover, iAs activated the ERK and JNK signaling pathways, which are involved in iAs-induced ROS production and genetic damage. Taken together, our present results suggest that elevated catalase activity in H-Ras(G12V)-transformed cells is significantly suppressed by iAs via activation of ERK and JNK signaling pathways and hence attenuate the defense of the neoplastic transformed cells against iAs-induced oxidative injuries.

Comparison of genome-wide DNA methylation in urothelial carcinomas of patients with and without arsenic exposure.

BACKGROUND: Arsenic is a well-documented carcinogen of human urothelial carcinoma (UC) with incompletely understood mechanisms. OBJECTIVES: This study aimed to compare the genome-wide DNA methylation profiles of arsenic-induced UC (AsUC) and non-arsenic-induced UC (Non-AsUC), and to assess associations between site-specific methylation levels and cumulative arsenic exposure. METHODS: Genome-wide DNA methylation profiles in 14 AsUC and 14 non-AsUC were analyzed by Illumina Infinium methylation27 BeadChip and validated by bisulfite pyrosequencing. Mean methylation levels (߯) in AsUC and non-AsUC were compared by their ratio (߯ ratio) and difference (Δ߯). Associations between site-specific methylation levels in UC and cumulative arsenic exposure were examined. RESULTS: Among 27,578 methylation sites analyzed, 231 sites had ߯ ratio >2 or <0.5 and 45 sites had Δ߯ >0.2 or <-0.2. There were 13 sites showing statistically significant (q<0.05) differences in ߯ between AsUC and non-AsUC including 12 hypermethylation sites in AsUC and only one hypermethylation site in non-AsUC. Significant associations between cumulative arsenic exposure and DNA methylation levels of 28 patients were observed in nine CpG sites of nine gens including PDGFD (Spearman rank correlation, 0.54), CTNNA2 (0.48), KCNK17 (0.52), PCDHB2 (0.57), ZNF132 (0.48), DCDC2 (0.48), KLK7 (0.48), FBXO39 (0.49), and NPY2R (0.45). These associations remained statistically significant for CpG sites in CTNNA2, KLK7, NPY2R, ZNF132 and KCNK17 in 20 non-smoking women after adjustment for tumor stage and age. CONCLUSIONS: Significant associations between cumulative arsenic exposure and methylation level of CTNNA2, KLK7, NPY2R, ZNF132 and KCNK17 were found in smoking-unrelated urothelial carcinoma. Arsenic exposure may cause urothelial carcinomas through the hypermethylation of genes involved in cell adhesion, proteolysis, transcriptional regulation, neuronal pathway, and ion transport. The findings of this study, which are limited by its small sample size and moderate dose-response relation, remain to be validated by further studies with large sample sizes.

Long-term low-dose exposure of human urothelial cells to sodium arsenite activates lipocalin-2 via promoter hypomethylation.

We previously reported that the sustained exposure of human urothelial cells (HUCs) to low-dose sodium arsenite induces changes in the gene expression profile and neoplastic transformation. In this study, we used the HumanMethylation27 BeadChip to analyze genome-wide methylation profiles and 5-aza-2'-deoxycytidine to examine the involvement of promoter methylation in gene expression. Because the expression of lipocalin-2 (LCN2) was highly enhanced by promoter hypomethylation in inorganic arsenic (iAs)-HUCs cells as well as bladder cancer tissues, we further showed that mutations at the binding sequences for NF-κB and C/EBP-α significantly reduced LCN2 promoter activity. By chromatin immunoprecipitation assay, we demonstrated the significantly increased binding of RelA (p65) and NF-κB1 (p50) to the hypomethylated promoter of LCN2 in the iAs-HUCs. Furthermore, we also demonstrated that LCN2 overexpression was crucial for the neoplastic characteristics of the iAs-HUCs, such as enhanced anchorage-independent growth, resistance to serum deprivation and activation of NF-κB signaling. In addition, our results indicated that enhanced NF-κB activity in iAs-HUCs was via LCN2-mediated increase in intracellular iron and reactive oxygen species levels. Taken together, our results show that sustained low-dose arsenic exposure results in epigenetic changes and enhanced oncogenic potential via LCN2 overexpression.

HSP70 colocalizes with PLK1 at the centrosome and disturbs spindle dynamics in cells arrested in mitosis by arsenic trioxide.

Heat shock protein 70 (HSP70) has been shown to be a substrate of Polo-like kinase 1 (PLK1), and it prevents cells arrested in mitosis by arsenic trioxide (ATO) from dying. Here, we report that HSP70 participates in ATO-induced spindle elongation, which interferes with mitosis progression. Our results demonstrate that HSP70 and PLK1 colocalize at the centrosome in ATO-arrested mitotic cells. HSP70 located at the centrosome was found to be phosphorylated by PLK1 at Ser6³¹ and Ser6³³. Moreover, unlike wild-type HSP70 (HSP70(wt)) and its phosphomimetic mutant (HSP70(SS631,633DD)), a phosphorylation-resistant mutant of HSP70 (HSP70(SS631,633AA)) failed to localize at the centrosome. ATO-induced spindle elongation was abolished in cells overexpressing HSP70(SS631,633AA). Conversely, mitotic spindles in cells ectopically expressing HSP70(SS631,633DD) were more resistant to nocodazole-induced depolymerization than in those expressing HSP70(wt) or HSP70(SS631,633AA). In addition, inhibition of PLK1 significantly reduced HSP70 phosphorylation and induced early onset of apoptosis in ATO-arrested mitotic cells. Taken together, our results indicate that PLK1-mediated phosphorylation and centrosomal localization of HSP70 may interfere with spindle dynamics and prevent apoptosis of ATO-arrested mitotic cells.

IFIT2 Depletion Promotes Cancer Stem Cell-like Phenotypes in Oral Cancer.

(1) Background: Cancer stem cells (CSCs) are a small cell population associated with chemoresistance, metastasis and increased mortality rate in oral cancer. Interferon-induced proteins with tetratricopeptide repeats 2 (IFIT2) depletion results in epithelial to mesenchymal transition, invasion, metastasis, and chemoresistance in oral cancer. To date, no study has demonstrated the effect of IFIT2 depletion on the CSC-like phenotype in oral cancer cells. (2) Methods: Q-PCR, sphere formation, Hoechst 33,342 dye exclusion, immunofluorescence staining, and flow cytometry assays were performed to evaluate the expression of the CSC markers in IFIT2-depleted cells. A tumorigenicity assay was adopted to assess the tumor formation ability. Immunohistochemical staining was used to examine the protein levels of IFIT2 and CD24 in oral cancer patients. (3) Results: The cultured IFIT2 knockdown cells exhibited an overexpression of ABCG2 and CD44 and a downregulation of CD24 and gave rise to CSC-like phenotypes. Clinically, there was a positive correlation between IFIT2 and CD24 in the patients. IFIT2high/CD24high/CD44low expression profiles predicted a better prognosis in HNC, including oral cancer. The TNF-α blockade abolished the IFIT2 depletion-induced sphere formation, indicating that TNF-α may be involved in the CSC-like phenotypes in oral cancer. (4) Conclusions: The present study demonstrates that IFIT2 depletion promotes CSC-like phenotypes in oral cancer.

The novel DNA alkylating agent BO-1090 suppresses the growth of human oral cavity cancer in xenografted and orthotopic mouse models.

Oral cancer is the fourth-most common cause of death in males and overall the sixth-most common cause of cancer death in Taiwan. Surgery, radiotherapy and chemotherapy combined with other therapies are the most common treatments for oral cavity cancer. Although cisplatin, 5-fluorouracil and docetaxel are commonly used clinically, there is no drug specific for oral cavity cancer. Here, we demonstrated that derivatives of 3a-aza-cyclopenta[a]indene, a class of newly synthesized alkylating agents, may be drugs more specific for oral cancer based on its potent in vitro cytotoxicity to oral cancer cells and on in vivo xenografts. Among them, BO-1090, bis(hydroxymethyl)-3a-aza-cyclopenta[a]indene derivative, targeted DNA for its cytotoxic effects as shown by inhibition of DNA synthesis (bromodeoxyuridine-based DNA synthesis assay), induction of DNA crosslinking (alkaline gel shift assay), and induction of DNA single-stranded breaks (Comet assay) and double-stranded breaks (γ-H2AX focus formation). Following DNA damage, BO-1090 induced G1/S-phase arrest and apoptosis in oral cancer cell lines. The therapeutic potential of BO-1090 was tested in mice that received a xenograft of oral cavity cancer cell lines (SAS or Cal 27 cells). Intravenous injection of BO-1090 significantly suppressed tumor growth in comparison to control mice. BO-1090 also significantly reduced the tumor burden in orthotopic mouse models using SAS cells. There was no significant adverse effect of BO-1090 treatment with this dosage based on whole blood count, biochemical enzyme profiles in plasma and histopathology of various organs in mouse. Taken together, our current results demonstrate that B0-1090 may have potential as a treatment for oral cavity cancer.

Synthesis of tunable and multifunctional Ni-doped near-infrared QDs for cancer cell targeting and cellular sorting.

Here, we report the facile preparation of tunable magnetic Ni-doped near-infrared (NIR) quantum dots (MNIR-QDs) as an efficient probe for targeting, imaging, and cellular sorting applications. We synthesized the MNIR-QDs via a hot colloidal synthesis approach to yield monodisperse and tunable QDs. These hydrophobic QDs were structurally and compositionally characterized and further functionalized with amino-PEG and carboxyl-PEG to improve their biocompatibility. Since QDs are known to be toxic due to the presence of cadmium, we have evaluated the in vitro and in vivo toxicity of our surface-functionalized MNIR-QDs. Our results revealed that surface-functionalized MNIR-QDs did not exhibit significant toxicity at the concentrations used in the experiments and are therefore suitable for biological applications. For further in vitro applications, we covalently linked folic acid to the surface of amino-PEG-coated MNIR-QDs through NHS chemistry to target the folate receptors largely present in the HeLa cells to demonstrate the specific targeting and magnetic behavior of these MNIR-QDs. Improved specificity has been observed with treatment of HeLa cells with the folic acid-linked amino PEG-coated MNIR QDs (FA-PEG-MNIR-QDs) compared to the one without folic acid. Since the synthesized probe has magnetic property, we have also successfully demonstrated sorting between the cells which have taken up the probe with the use of a magnet. Our findings strongly suggest that these functionalized MNIR-QDs can be a potential probe for targeting, cellular sorting, and bioimaging applications.

Development of chitosan oligosaccharide-modified gold nanorods for in vivo targeted delivery and noninvasive imaging by NIR irradiation.

In the present study, we demonstrate the synthesis and applications of multifunctional gold nanorod-based probes for specific targeting and noninvasive imaging based on localized heating generated by gold nanorods after NIR irradiation. The structural design of the probe consists of MUA (11-mercaptoundecanoic acid)-capped gold nanorods covalently linked with low-molecular-weight chitosan oligosaccharide (M(w) ~5000) via carbodiimide (EDC) coupling agent. This surface modification is performed for complete replacement of toxic CTAB (hexadecyltrimethyl-ammonium chloride) and acid-responsive delivery of gold nanorods in acidic environment as known to be present at tumor surrounding areas. The resulting chitosan oligosaccharide-modified gold nanorods (CO-GNRs) were further conjugated with tumor targeting monoclonal antibody against EGFR (epidermal growth factor receptor) to provide localized targeting functionality owing to the overexpression of EGFR in human oral adenosquamous carcinoma cell line CAL 27. Initial in vitro and in vivo toxicity assessments indicated that CO-GNRs did not induce any significant toxicity and are thus suitable for biological applications. Furthermore, selective targeting and accumulation of CO-GNRs were observed in vitro via two-photon luminescence imaging studies in CAL 27, which was also observed through in vivo targeting studies performed via NIR (near-infrared) laser irradiation in CAL 27 xenografts of BALB/c nude mice. Hence, the CO-GNRs that we have developed are biocompatible and nontoxic and can be a potential candidate for in vivo targeted delivery, noninvasive imaging based on localized hyperthermia, and photothermal-related therapies.

Proteomic identification of Hsp70 as a new Plk1 substrate in arsenic trioxide-induced mitotically arrested cells.

We previously demonstrated that when arsenic trioxide (ATO)-induced mitotically arrested HeLa S3 cells (AIMACs) were treated with staurosporine (SSP) the cells rapidly exited mitosis. To better define the cellular targets and the underlying mechanisms of AIMACs, we applied 2-D DIGE followed by LC-MS/MS analysis and showed that SSP induced a significant change in the phosphoproteome of AIMACs. Among the proteins whose phosphorylation was modulated by SSP, we identified Hsp70, Rad 23B, and eukaryotic translation initiation factor 4B as potentially new substrates of polo-like kinase 1 (Plk1), an essential serine/threonine kinase with versatile mitotic functions. Since Hsp70 is a stress protein responsible for ATO treatment, we further identified Thr(13) , Ser(362) , Ser(631) , and Ser(633) on Hsp70 intracellularly phosphorylated in AIMACs by combining TiO(2) phospho-peptides enrichment and MS/MS analysis. Using antibody specifically against phosph-Ser(631) Hsp70 and further aided by expression of kinase-dead Plk1 and pharmacological inhibition of Plk1, we concluded that Ser(631) on Hsp70 is phosphorylated by Plk1 in AIMACs. By immnuofluorescent staining, we found the colocalization of Hsp70 and Plk1 in AIMACs but not in interphase cells. In addition, Plk1-mediated phosphorylation of Hsp70 prevented AIMACs from mitotic death. Our results reveal that Hsp70 is a novel substrate of Plk1 and that its phosphorylation contributes to attenuation of ATO-induced mitotic abnormalities.

Cytotoxicity of arsenic trioxide is enhanced by (-)-epigallocatechin-3-gallate via suppression of ferritin in cancer cells.

Arsenic trioxide (ATO) treatment is a useful therapy against human acute promyelocytic leukemia (APL), however, it concomitantly brings potential adverse consequences including serious side effect, human carcinogenicity and possible development of resistance. This investigation revealed that those problems might be relaxed by simultaneous application with (-)-epigallocatechin-3-gallate (EGCG), one of the major components from green tea. EGCG significantly lowered down the ATO concentration required for an effective control of APL cells, HL-60. The simultaneous treatment of ATO with EGCG induced a mitochondria-dependent apoptosis in HL-60 cells significantly, which accounted for more than 70% of the cell death in the treatment. The mechanism of apoptosis induction was elucidated. EGCG in HL-60 cells acted as a pro-oxidant enhancing intracellular hydrogen peroxide significantly. ATO, on the other hand, induced heme oxygenase-1 (HO-1) to catalyze heme degradation, thereby provided ferrous iron for EGCG-induced hydrogen peroxide to precede Fenton reaction, which in turn generated deleterious reactive oxygen species to damage cell. In addition, EGCG inhibited expression of ferritin, which supposedly to sequester harmful ferrous iron, thereby augmented the occurrence of Fenton reaction. This investigation also provided evidence that ATO, since mainly acted to induce HO-1 in simultaneous treatment with EGCG, could be replaced by other HO-1 inducer with much less human toxicity. Furthermore, several of our preliminary investigations revealed that the enhanced cytotoxicity induced by combining heme degradation and Fenton reaction is selectively toxic to malignant but not non-malignant cells.

Enhanced suppression of tumor growth by concomitant treatment of human lung cancer cells with suberoylanilide hydroxamic acid and arsenic trioxide.

The efficacy of arsenic trioxide (ATO) against acute promyelocytic leukemia (APL) and relapsed APL has been well documented. ATO may cause DNA damage by generating reactive oxygen intermediates. Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, modulates gene and protein expression via histone-dependent or -independent pathways that may result in chromatin decondensation, cell cycle arrest, differentiation, and apoptosis. We investigated whether ATO and SAHA act synergistically to enhance the death of cancer cells. Our current findings showed that combined treatment with ATO and SAHA resulted in enhanced suppression of non-small-cell lung carcinoma in vitro in H1299 cells and in vivo in a xenograft mouse model. Flow cytometric analysis of annexin V+ cells showed that apoptotic cell death was significantly enhanced after combined treatment with ATO and SAHA. At the doses used, ATO did not interfere with cell cycle progression, but SAHA induced p21 expression and led to G1 arrest. A Comet assay demonstrated that ATO, but not SAHA, induced DNA strand breaks in H1299 cells; however, co-treatment with SAHA significantly increased ATO-induced DNA damage. Moreover, SAHA enhanced acetylation of histone H3 and sensitized genomic DNA to DNase I digestion. Our results suggest that SAHA may cause chromatin relaxation and increase cellular susceptibility to ATO-induced DNA damage. Combined administration of SAHA and ATO may be an effective approach to the treatment of lung cancer.

Novel bifunctional alkylating agents, 5,10-dihydropyrrolo[1,2-b]isoquinoline derivatives, synthesis and biological activity.

A series of linear pyrrolo[1,2-b]isoquinoline derivatives was synthesized for antitumor evaluation. The preliminary antitumor studies reveal that both bis(hydroxymethyl) and their bis(alkylcarbamate) derivatives show significant antitumor activity in inhibiting various human tumor cell growth in vitro. 1,2-Bis(hydroxymethyl)-3-methyl-5,10-dihydropyrrolo[1,2-b]isoquinoline (20a) was selected for antitumor studies in animal models. The results show that this agent can induce complete tumor remission or significant suppression in nude mice bearing human breast (MX-1) xenograft and ovarian (SK-OV-3) xenografts, respectively. Alkaline agarose gel shifting assay showed that 20a is able to cross-link with DNA. Studies on the cell cycle inhibition revealed that this agent induces cell arrest at G2/M phase. The results warrant further antitumor investigation against other human tumor growth in animal models.

Design, synthesis and antitumor evaluation of phenyl N-mustard-quinazoline conjugates.

A series of N-mustard-quinazoline conjugates was synthesized and subjected to antitumor studies. The N-mustard pharmacophore was attached at the C-6 of the 4-anilinoquinazolines via a urea linker. To study the structure-activity relationships of these conjugates, various substituents were introduced to the C-4 anilino moiety. The preliminary antitumor studies revealed that these agents exhibited significant antitumor activity in inhibiting various human tumor cell growths in vitro. Compounds 21b, 21g, and 21h were selected for further antitumor activity evaluation against human breast carcinoma MX-1 and prostate PC-3 xenograft in animal model. These agents showed 54-75% tumor suppression with low toxicity (5-7% body-weight changes). We also demonstrate that the newly synthesized compounds are able to induce DNA cross-linking through alkaline agarose gel shift assay and inhibited cell cycle arrest at G2/M phase.

Design, synthesis, and biological evaluation of novel water-soluble N-mustards as potential anticancer agents.

A series of novel water-soluble N-mustard-benzene conjugates bearing a urea linker were synthesized. The benzene moiety contains various hydrophilic side chains are linked to the meta- or para-position of the urea linker via a carboxamide or an ether linkage. The preliminary antitumor studies revealed that these agents exhibited potent cytotoxicity in vitro and therapeutic efficacy against human tumor xenografts in vivo. Remarkably, complete tumor remission in nude mice bearing human breast carcinoma MX-1 xenograft and significant suppression against prostate adenocarcinoma PC3 xenograft were achieved by treating with compound 9aa' at the maximum tolerable dose with relatively low toxicity. We also demonstrate that the newly synthesized compounds are able to induce DNA cross-linking through alkaline agarose gel shift assay. A pharmacokinetic profile of the representative 9aa' in rats was also investigated. The current studies suggest that this agent is a promising candidate for preclinical studies.

Discovery of Oral Anticancer 1,2-Bis(hydroxymethyl)benzo[g]pyrrolo[2,1-a]phthalazine Hybrids That Inhibit Angiogenesis and Induce DNA Cross-Links.

Designing hybrid molecules with dual functions is one approach to improve the therapeutic efficacy of combination treatment. We have previously conjugated phthalazine and bis(hydroxymethyl)pyrrole pharmacophores to form hybrids bearing antiangiogenesis and DNA interstrand cross-linking activities. To improve the bioavailability, we adopted a benzology approach to design and synthesize a new series of 1,2-bis(hydroxymethyl)benzo[g]pyrrolo[2,1-a]phthalazines. These new hybrids retained the dual functions and could be formulated into vehicles for intravenous and oral administration. Among them, we demonstrated that compound 19a with dimethylamine at the C6 position markedly suppressed the tumor growth of human small cell lung cancer cell line H526, squamous lung cancer cell line H520, and renal cancer cell line 786-O in nude mice, implying that compound 19a is a broad-spectrum anticancer agent. Our results implicated that the conjugation of antiangiogenic and DNA cross-linking is likely to be a helpful approach to improving the efficacy of combination therapy.

GT-Repeat Polymorphism in the HO-1 Gene Promoter Is Associated with Risk of Liver Cancer: A Follow-Up Study from Arseniasis-Endemic Areas in Taiwan.

The induction of heme oxygenase-1 (HO-1) has been shown to have therapeutic potential in experimental models of hepatitis and liver fibrosis, which are closely related to liver cancer. In humans, HO-1 induction is transcriptionally modulated by the length of a GT-repeat [(GT)n] in the promoter region. We aimed to investigate the effect of HO-1 (GT)n variants on liver cancer in a human population. We determined the HO-1 genotype in 1153 study subjects and examined their association with liver cancer risk during a 15.9-year follow-up. Allelic polymorphisms were classified as short [S, <27 (GT)n] or long [L, ≥27 (GT)n]. Newly developed cancer cases were identified through linkage to the National Cancer Registry of Taiwan. Multivariate Cox regression analysis was used to evaluate the effect of the HO-1 (GT)n variants. Alpha-fetoprotein (AFP) and cirrhosis history were also examined. The S/S genotype was found to be significantly associated with liver cancer risk, compared to the L/S and L/L genotypes. The S/S genotype group also had a higher percentage of subjects with abnormal AFP levels than other groups. There were significant percentages of cirrhosis among groups who carried S-alleles. Our findings indicate that short (GT)n variants in the HO-1 gene may confer susceptibility to rather than protection from liver cirrhosis/cancer.