Abstract. Oral squamous cell carcinoma (OSCC) is a prevalent cancer worldwide. Let-7 family has been shown to function as a tumor suppressor through regulating multiple oncogenic signaling. Recent study reported that combined underexpression of miR-205 and let-7d showed negative correlation with the survival prognosis of head and neck cancer patients. However, the let-7d-involved mechanism in regulating OSCC is still unclear. In this study, we first demonstrated that let-7d expression was significantly decreased while Twist and Snail expression was increased in OSCC cancer cell lines and primary cultures as compared to normal human oral keratinocyte cells. To further investigate the role of let-7d in OSCC, we applied the SPONGE method to knock down let-7d in OECM-1 and two primary OSCC cell types. The results showed that knockdown of let-7d promote epithelialmesenchymal transition (EMT) traits and migratory/invasive capabilities in OSCC cells. Furthermore, down-expression of let-7d significantly activated Twist and Snail expressions and chemo-resistant abilities of OSCC cells. Notably, overexpression of let-7d effectively reversed the EMT phenotype, blocked migratory/invasive abilities, and further increased the chemosensitivity in oral cancer tumor initiating ALDH1 + cells. In sum, these results show that let-7d negatively modulates EMT expression and also plays a role in regulating chemo-resistant ability in oral cancer. IntroductionHead and neck squamous cell carcinoma, including oral squamous cell carcinoma (OSCC), is the sixth most prevalent malignancy worldwide and accounts for approximately 8-10% of all cancers in Southeast Asia (1,2). The prognosis of OSCC remains dismal as more than 50% of the patients die of this disease or complications within 5 years under current therapies (2). To increase patient survival rate, investigations elucidating the mechanisms of tumorigenesis in OSCC are urgently needed (2). Some studies have suggested that subsets of cancer stem cells (CSC) or tumor initiating cells (TICs) are responsible for tumor progression as well as recurrence after conventional chemotherapy (3). However, there is lack of suitable markers for isolating the crucial subset of tumor cells that is capable of reforming new tumors in vivo and accounts for tumor relapse in OSCC, according to CSC hypothesis of tumorigenesis.MicroRNAs (miRNAs), highly conserved small RNA molecules that regulate gene expression, can act as cancer signatures, oncogenes or tumor suppressors (4). The ubiquitously expressed let-7/miR-98 family was one of the first mammalian miRNAs to be identified (5-8). Let-7 family members have been described as being down-regulated during cancer progression in various human cancers including lung, gastric, ovarian, colon cancer, leiomyoma and melanoma (5-13). Let-7d, a member of the let-7 family of miRNAs, has also been shown to act as tumor suppressor, most likely through targeting RAS (14) or high mobility group A2 (15). Let-7d regulates senescence of human cord blood-derived multipotent ste...

Cantharidin is found in Mylabris caraganae and various other insects and shows extremely high vesicant potency and toxic properties. [1][2][3][4] In a pharmacologic study, cantharidin increased the force of contraction in isolated guinea pig papillary muscles.5) It can also cause severe congestion and edema of the liver.6) Recent reports have indicated that the action of cantharidin in mice is associated with binding to protein phosphatase 2A in liver cytosol and inhibition of its phosphorylase phosphatase activity. 7,8) In clinical studies it had significant activity against liver tumors and the KB cell line in tissue culture at low concentrations.9-12) In our previous studies,2) increasing the solubility of the title compounds decreased the toxicity while maintaining the biological activities. N-Thiazolyl and thiadiazolyl cantharidinimides with better solubility showed cytotoxicity against human hepatocellular carcinoma cell lines, which encouraged us to prepare cantharidinimide derivatives. These compounds were synthesized from cantharidin and primary amines in triethylamine by heating to ca. 200°C. N-Methylcantharidinimide has shown tumor-inhibitory action in animals, and some cantharidinimides were tested for their effects on xanthine oxidase; those tests showed inhibitory effects on xanthine oxidase.13) Some cantharidinimides also show antiplatelet effects on thrombin, arachidonic acid, collagen, and platelet-activating factor-induced aggregation.Chemistry We prepared effective cantharidinimides by heating the reactants cantharidin 1 and primary amines, aniline derivatives, and aminopyridines to ca. 200°C with 3 ml of dry toluene and 1-2 ml of triethylamine in a high-pressure sealed tube (Buchi glasuster 0032) to provide cantharidinimides (compounds 2-22, Chart 1) in good yields. In further studies, all of the synthetic compounds were tested for their cytotoxicity in suppressing the growth of the human carcinoma cell lines HL-60 and Hep G2. Results and DiscussionWith the high-pressure technique, we obtained cantharidinimides (after recrystallization from methanol) in yields of 29-96%, as listed in Table 1. The highest yields were obtained from aliphatic primary amines, (2-4) as well as phenetyl amines 18-22. The variable yields of compounds 5-16, which had parent aniline and pyridine components, might reflect the inductive electron negative effect influencing the yield. The results of these yields strongly confirm the influence of amine nucleophilicity and basicity. The potential cytotoxicities of the prepared cantharidinimides were investigated against hepatocellular carcinoma cell (Hep G2) and human myeloid leukemia cell (HL-60) lines and evaluated Modification of the cantharidinimide structure led to the discovery of a novel class of antitumor compounds. These cantharidinimide derivatives containing aliphartic, aryl, and pyridyl groups showed some effect in vitro against HepG2 and HL-60 cells.

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As a part of our program aimed at exploring the biological activity of symmetrical substitution of side chains into the anthracene-9,10-dione chromophore, we have synthesized a series of 1,5-bisthioanthraquinones 2 and 1,5-bisacyloxyanthraquinones 3 that are related to the antitumor agent mitoxantrone. Since the telomerase enzyme is a novel target for potential anticancer therapy and stem cell expansion, we explore the biological effects of these compounds by evaluating their effects on telomerase activity and telomerase expression. Telomerase is required for telomere maintenance and is active in most human cancers and in germinal cells but not in most of the normal human somatic tissues. We found that most of the 1,5-disubstituted anthraquinones did not exhibit inhibitory activity at the concentration ranging from 20 to 30 microM. To facilitate the analysis of the expression of telomerase, we used cancer and normal cell lines that carry secreted alkaline phosphatase (SEAP) gene under the control of human telomerase reverse transcriptase (hTERT). The effects of these compounds on the expression of telomerease were analyzed using the cell-based reporter systems. While most of these compounds did not appear to selectively repress the expression of hTERT in cancer cells, compounds 3a, 3d, and 3i activated hTERT expression in normal cells. The effects of these three compounds on hTERT expression appear to be specific because they did not increase the expression of a CMV promoter-driven SEAP. Thus, in addition to anticancer functions, our finding raises the possibility that these compounds might also have a role in cell immortalization. The application of these anthraquinone derivatives in stem cell research and tissue engineering is also discussed.

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