ABCG2, CD44 and SOX9 are increased with the acquisition of drug resistance and involved in cancer stem cell activities in head and neck squamous cell carcinoma cells.

Cancer stem cells are a sub-population of cancer cells with self-renewal activity that play key roles in tumor resistance to chemotherapy and radiation. Several cancer stem cell markers have been identified to correlate with clinical prognosis. However, which marker is associated with which cancer stem cell characteristic is unclear. The present study aimed to clarify the relationship between cancer stem cell markers associated with drug resistance acquisition and the characteristics of cancer stem cells. We generated cisplatin-resistant head and neck squamous cell carcinoma cells by culturing cells in increasing concentrations of cisplatin. The cisplatin-resistant head and neck squamous cell carcinoma cells also acquired multidrug resistance and were named resistant HSC-3 (R HSC-3) cells. R HSC-3 showed no differences in cell proliferation or cell cycle distributions compared with parental cells. R HSC-3 cells showed increased drug excretion ability and elevated expression of ATP-binding cassette subfamily G member 2 (ABCG2), a drug excretion pump. R HSC-3 cells also highly expressed CD44, a cancer stem cell marker, and exhibited enhanced cell invasion and spheroid formation abilities. Furthermore, the stem cell-related factor SRY-box transcription factor 9 (SOX9) was identified as increased in R HSC-3 cells by microarray analysis. Knockdown experiments showed that SOX9 and ABCG2 were involved in the drug excretion ability of R HSC3 cells and ABCG2 was involved in the spheroid formation ability of R HSC-3 cells. These results indicate that CD44, SOX9 and ABCG2 expression levels were enhanced in head and neck squamous cell carcinoma cells that acquired multidrug resistance and that these molecules are important for maintaining cancer stem cell characteristics. Overall, regulating CD44, SOX9 and ABCG2 may be a strategy to inhibit cancer stem cells.

Bioactivity inspired C19-diterpenoid alkaloids for overcoming multidrug-resistant cancer.

The pharmacological activities of C19-diterpenoid alkaloids are related to their basic skeletons (e.g., aconitine-type or lycoctonine-type). Also, few studies have been reported on the chemosensitizing effects of diterpenoid alkaloids. Consequently, this study was aimed at determining the chemosensitizing effects of synthetic derivatives of lycoctonine-type C19-diterpenoid alkaloids on a P-glycoprotein (P-gp)-overexpressing multidrug-resistant (MDR) cancer cell line KB-VIN. The acyl-derivatives of delpheline and delcosine showed moderate cytotoxicity against chemosensitive cancer cell lines. Among non-cytotoxic synthetic analogs (1-14), several derivatives effectively and significantly sensitized MDR cells by interfering with the drug transport function of P-gp to three anticancer drugs, vincristine, paclitaxel, and doxorubicin. The chemosensitizing effect of derivatives 2, 4, and 6 on KB-VIN cells against vincristine were more potent than 5 µM verapamil, and derivatives 4 and 13 were more effective than 5 µM verapamil for paclitaxel. Among them, 2 in particular increased the sensitivity of KB-VIN cells to vincristine by 253-fold.

Shikonin induces odontoblastic differentiation of dental pulp stem cells via AKT-mTOR signaling in the presence of CD44.

Purpose: In our previous study, we demonstrated that hyaluronan induces odontoblastic differentiation of dental pulp stem cells via interactions with CD44. However, it remains unclear whether CD44 expression by dental pulp stem cells is required for odontoblastic differentiation.Methods: We searched for a compound other than hyaluronan that induces odontoblastic differentiation of dental pulp stem cells and used western blotting to determine whether CD44 is involved in the induction of odontoblastic differentiation by the compound. We further validated the cell signaling details of the compound-induced expression of dentin sialophosphoprotein (DSPP), which is known as a marker of odontoblastic differentiation.Results: We investigated shikonin, which is one of the derivatives of naphthoquinone, the skeleton of vitamin K. Shikonin-induced expression of DSPP was inhibited by PI3K, AKT, and mTOR inhibitors. Additionally, shikonin-induced expression of DSPP was inhibited in dental pulp stem cells transfected with siRNA against CD44.Conclusions: Shikonin can stimulate dental pulp stem cells to undergo odontoblastic differentiation through a mechanism involving the AKT-mTOR signaling pathway and CD44. Although expression of CD44 is important for inducing odontoblastic differentiation of dental pulp stem cells, the relationship between the AKT-mTOR signaling pathway and CD44 expression, in the context of shikonin stimulation, has not yet been elucidated. This study suggests that shikonin may be useful for inducing odontoblastic differentiation of dental pulp stem cells, and that it may have clinical applications, including protection of the dental pulp.

Combined SN-38 and gefitinib treatment promotes CD44 degradation in head and neck squamous cell carcinoma cells.

The aim of the present study was to search for an effective regimen among existing chemotherapies for head and neck squamous cell carcinoma (HNSCC). Among the tested drugs, we focused on combined SN-38, which is the active metabolite produced from irinotecan hydrochloride - a type I DNA topoisomerase inhibitor - after it is metabolized by carboxylesterase in the liver and gefitinib, an EGFR tyrosine kinase inhibitor treatment, based on the ability of this combination to inhibit HNSCC cell growth. Contrary to our expectation, in vivo, there was no significant difference in tumor growth suppression between gefitinib-only treatment and gefitinib plus SN-38. However, when tumor measurements were continued after treatment ceased, we found that several tumors showed renewed growth in the gefitinib-only group. The tumors that resumed growing after treatment showed increased CD44 expression compared with tumors from the combined treatment group. Next, we investigated the mechanism whereby SN-38 and gefitinib inhibited CD44 expression in vitro. These studies revealed that the combined treatment promoted lysosomal degradation of CD44. The present study revealed that combined gefitinib and SN-38 treatment inhibits CD44 expression by promoting its lysosomal degradation in HNSCC cells. However, it is still unclear whether inhibition of CD44 expression in HNSCC cells can directly suppress tumor regrowth after therapy. Thus, it may be necessary to elucidate the relationship between the effects of these chemotherapeutic agents on CD44 expression and tumor recurrence/metastasis in future studies.

Metastasized murine oral squamous cell carcinoma cells induce intratumoral polymorphonuclear myeloid derived suppressor cells.

Myeloid derived suppressor cells (MDSCs) localize to hematopoietic organs and peripheral blood during inflammation or tumor tissues and lymph nodes in the presence of a tumor. However, whether there are differences in MDSCs found in the primary tumor and metastases is unknown. In the present study, we established a cell line of metastasized tumor cells to a lymph node, L5-11, which were derived from the Sq-1979 mouse buccal mucosa squamous cell carcinoma cell line. We then analyzed tumor immunogenicity, especially with regard to MDSCs, to clarify the differences between the primary tumor and metastases, using an isogenic heterotopic tumor transplantation model. Our data showed that the population of intratumoral MDSCs, especially polymorphonuclear MDSCs in the lymph node metastasis model were significantly increased compared with syngeneic grafts from the primary cell line Sq-1979 after 21 days. Furthermore, we identified that the lymph node metastasis cell line had increased expression of genes that promote the expansion of MDSCs, tumor growth and metastasis. Hence, these data suggest that tumor immunosuppression can occur via activation of MDSCs. However, further examination is required to clarify whether all or a subset of these factors from the lymph node metastasis tumor cells are required to induce intratumoral MDSCs.

Induced overexpression of CD44 associated with resistance to apoptosis on DNA damage response in human head and neck squamous cell carcinoma cells.

CD44 is a marker of cancer stem cells in head and neck squamous cell carcinoma, and CD44 expression is related to prognosis in cancer patients. We examined whether herbal medicine components affect CD44 expression and induce cancer cell apoptosis. Baicalin enhanced apoptosis with no effect on CD44 levels, while baicalein did not enhance apoptosis and upregulated CD44 in head and neck squamous cell carcinoma. Furthermore, baicalein induced phosphorylation of CHK1, as a marker of DNA damage response to S-to-G2/M phase arrest. Our results clearly demonstrated that baicalein enhanced expression of CD44 and accordingly enhanced the DNA damage response. These data suggest that induction of CD44 inhibited cancer cell induction of apoptosis by increasing the DNA damage response. Together, our findings suggest that CD44 expression in head and neck squamous cell carcinoma plays a role in enhancing the DNA damage response.

Triethylated chromones with substituted naphthalenes as tubulin inhibitors.

Previously synthesized 2-(benzo[b]thiophene-3'-yl)-6,8,8-triethyldesmosdumotin B (1, TEDB-TB) and 2-(naphth-1'-yl)-6,8,8-triethyldesmosdumotin B (2) showed potent activity against multiple human tumor cell lines, including a multidrug-resistant (MDR) subline, by targeting spindle formation and/or the microtubule network. Consequently, ester analogues of hydroxylated naphthyl substituted TEBDs (3-5) were prepared and evaluated for their effects on tumor cell proliferation and on tubulin assembly. Among all new compounds, compound 6, a 4'-acetoxynaphthalen-1'-yl derivative, displayed the most potent antiproliferative activity (IC50 0.2-5.7µM). Selected analogues were confirmed to be tubulin assembly inhibitors in cell-free and cell-based assays using MDR tumor cells. The new analogues partially inhibited colchicine binding to tubulin, suggesting their binding mode would be different from that of colchicine. This observation was supported by computational docking model analyses. Thus, the newly synthesized triethylated chromones with esterified naphthalene groups have good potential for development as a new class of mitotic inhibitors that target tubulin.

Synthesis, biological evaluation, and physicochemical property assessment of 4-substituted 2-phenylaminoquinazolines as Mer tyrosine kinase inhibitors.

Current results identified 4-substituted 2-phenylaminoquinazoline compounds as novel Mer tyrosine kinase (Mer TK) inhibitors with a new scaffold. Twenty-one 2,4-disubstituted quinazolines (series 4-7) were designed, synthesized, and evaluated against Mer TK and a panel of human tumor cell lines aimed at exploring new Mer TK inhibitors as novel potential antitumor agents. A new lead, 4b, was discovered with a good balance between high potency (IC50 0.68µM) in the Mer TK assay and antiproliferative activity against MV4-11 (GI50 8.54µM), as well as other human tumor cell lines (GI50<20µM), and a desirable druglike property profile with low logP value (2.54) and high aqueous solubility (95.6µg/mL). Molecular modeling elucidated an expected binding mode of 4b with Mer TK and necessary interactions between them, thus supporting the hypothesis that Mer TK might be a biologic target of this kind of new active compound.

Optimization of N-aryl-6-methoxy-1,2,3,4-tetrahydroquinolines as tubulin polymerization inhibitors.

Thirteen new N-aryl 1,2,3,4-tetrahydroquinoline compounds (4a-f, 6a-c, and 8a-d) were synthesized and evaluated for antitumor activity and drug-like properties. Compound 4a exhibited high inhibitory potency with low nanomolar GI50 values of 16-20 nM in cellular assays, including excellent activity against the P-glycoprotein overexpressing cell line KBvin. Compound 4a inhibited colchicine binding to tubulin and tubulin assembly with an IC50 value of 0.85 µM, superior to the reference compound CA4 (1.2 µM) in the same assay. In addition, 4a also exhibited highly improved water solubility (75 µg/mL) and a suitable logP value (3.43) at pH 7.4. With a good balance between antitumor potency and drug-like properties, compound 4a could be a new potential drug candidate for further development. Current results on SAR studies and molecular modeling provided more insight about this class of compounds as tubulin polymerization inhibitors targeting the colchicine site.

Evaluation of Aconitum diterpenoid alkaloids as antiproliferative agents.

Little information has been reported on the antitumor effects of the diterpenoid alkaloid constituents of Aconitum plants, used in the herbal drug 'bushi'. This study was aimed at determining the antitumor activities of Aconitum C19-and C20-diterpenoid alkaloids and synthetic derivatives against lung (A549), prostate (DU145), nasopharyngeal (KB), and vincristine-resistant nasopharyngeal (KB-VIN) cancer cell lines. Newly synthesized C20-diterpenoid alkaloid derivatives showed substantial suppressive effects against all human tumor cell lines tested. In contrast, natural and derivatized C19-diterpenoid alkaloids showed only a slight or no effect. Most of the active compounds were hetisine-type C20-diterpenoid alkaloids, specifically kobusine and pseudokobusine analogs with two different substitution patterns, C-11 and C-11,15. Notably, several C20-diterpenoid alkaloids were more potent against multidrug-resistant KB subline KB-VIN cells. Pseudokobusine 11-3'-trifluoromethylbenzoate (94) is a possible promising new lead meriting additional evaluation against multidrug-resistant tumors.

Development of a novel class of tubulin inhibitor from desmosdumotin B with a hydroxylated bicyclic B-ring.

A series of newly synthesized hydroxylated analogues of triethyldesmosdumotin B (TEDB) with a bicyclic B-ring exhibited a significantly different mode of action for affecting microtubule dynamics and spindle formation but had the same antiproliferative activity spectrum, including activity against multidrug-resistant tumors. These analogues efficiently induced cell cycle arrest at prometaphase and caused formation of immature multipolar spindles. 6'-Hydroxyl TEDB-TB (8) disrupted bipolar spindle formation but had a negligible effect on interphase microtubules. On the basis of the predicted binding modes of the new compounds with tubulin dimer, compound 4 forms three hydrogen bonds (H-bonds) only with α-tubulin at the colchicine site; in contrast, 8 forms H-bonds with both α- and ß-tubulin. We predict that, when a compound/ligand, such as 8, forms H-bonds to both α- and ß-tubulins, spindle formation is disrupted more than the dynamics of interphase microtubules. This result may reflect the well-known greater dynamicity of spindle microtubules as compared with interphase microtubules.

Optimization of 4-(N-cycloamino)phenylquinazolines as a novel class of tubulin-polymerization inhibitors targeting the colchicine site.

The 6-methoxy-1,2,3,4-tetrahydroquinoline moiety in prior leads 2-chloro- and 2-methyl-4-(6-methoxy-3,4-dihydroquinolin-1(2H)-yl)quinazoline (1a and 1b) was modified to produce 4-(N-cycloamino)quinazolines (4a-c and 5a-m). The new compounds were evaluated in cytotoxicity and tubulin inhibition assays, resulting in the discovery of new tubulin-polymerization inhibitors. 7-Methoxy-4-(2-methylquinazolin-4-yl)-3,4-dihydroquinoxalin- 2(1H)-one (5f), the most potent compound, exhibited high in vitro cytotoxic activity (GI50 1.9-3.2 nM), significant potency against tubulin assembly (IC50 0.77 µM), and substantial inhibition of colchicine binding (99% at 5 µM). In mechanism studies, 5f caused cell arrest in G2/M phase, disrupted microtubule formation, and competed mostly at the colchicine site on tubulin. Compound 5f and N-methylated analogue 5g were evaluated in nude mouse MCF7 xenograft models to validate their antitumor activity. Compound 5g displayed significant in vivo activity (tumor inhibitory rate 51%) at a dose of 4 mg/kg without obvious toxicity, whereas 5f unexpectedly resulted in toxicity and death at the same dose.

Discovery of novel antitumor dibenzocyclooctatetraene derivatives and related biphenyls as potent inhibitors of NF-κB signaling pathway.

Several dibenzocyclooctatetraene derivatives (5-7) and related biphenyls (8-11) were designed, synthesized, and evaluated for inhibition of cancer cell growth and the NF-κB signaling pathway. Compound 5a, a dibenzocyclooctatetraene succinimide, was discovered as a potent inhibitor of the NF-κB signaling pathway with significant antitumor activity against several human tumor cell lines (GI50 1.38-1.45 µM) and was more potent than paclitaxel against the drug-resistant KBvin cell line. Compound 5a also inhibited LPS-induced NF-κB activation in RAW264.7 cells with an IC50 value of 0.52 µM, prevented IκB-α degradation and p65 nuclear translocation, and suppressed LPS-induced NO production in a dose-dependent manner. The antitumor data in cellular assays indicated that relative positions and types of substituents on the dibenzocyclooctatetraene or acyclic biphenyl as well as torsional angles between the two phenyls are of primary importance to antitumor activity.

N-aryl-6-methoxy-1,2,3,4-tetrahydroquinolines: a novel class of antitumor agents targeting the colchicine site on tubulin.

Structural optimizations of the prior lead 1a led to the discovery of a series of N-aryl-6-methoxy-1,2,3,4-tetrahydroquinoline derivatives as a novel class of tubulin polymerization inhibitors targeted at the colchicine binding site. The most active compound 6d showed extremely high cytotoxicity against a human tumor cell line panel (A549, KB, KBvin, and DU145) with GI50 values ranging from 1.5 to 1.7 nM, significantly more potent than paclitaxel, especially against the drug-resistant KBvin cell line, in the same assays. Analogs 5f, 6b, 6c, and 6e were also quite potent, with a GI50 range of 0.011-0.19 µM. In further studies, active compounds 6b-e and 5f significantly inhibited tubulin assembly, with IC50 values of 0.92-1.0 µM and strongly inhibited colchicine binding to tubulin, with inhibition rates of 75-99% (at 5 µM), comparable with or more potent than combretastatin A-4 (IC50 0.96 µM). Current studies included design, synthesis, and biological evaluations of 24 new compounds (series 3-6). Related SAR analysis, molecular modeling, and evaluation of essential drug-like properties, i.e. water solubility, log P, and in vitro metabolic stability, were also performed.

Discovery of novel andrographolide derivatives as cytotoxic agents.

The natural diterpenoid andrographolide (1) exhibits various biological activities. Seventeen derivatives of 1 were prepared via esterification and etherification of 14-dehydroxy-11,12-didehydroandrographolide (2). Most derivatives demonstrated significant inhibition against tumor cell growth. The most active compounds, 3b and 3c, had GI50 values of 1.46-9.19 µM against A549, DU145, KB and KB-Vin tumor cells. In an immunocytochemical study, treatment with compound 3c disrupted microtubule dynamics in PC-3 cells, but caused no accumulation of metaphase cells, which is a phenotype dissimilar from that of 1. This difference suggests that structural modification of 1 resulted in a shift in the underlying molecular mechanism.

Synthesis and biological evaluation of N-alkyl-N-(4-methoxyphenyl)pyridin-2-amines as a new class of tubulin polymerization inhibitors.

Based on our prior antitumor hits, 32 novel N-alkyl-N-substituted phenylpyridin-2-amine derivatives were designed, synthesized and evaluated for cytotoxic activity against A549, KB, KB(VIN), and DU145 human tumor cell lines (HTCL). Subsequently, three new leads (6a, 7g, and 8c) with submicromolar GI(50) values of 0.19-0.41 µM in the cellular assays were discovered, and these compounds also significantly inhibited tubulin assembly (IC(50) 1.4-1.7 µM) and competitively inhibited colchicine binding to tubulin with effects similar to those of the clinical candidate CA-4 in the same assays. These promising results indicate that these tertiary diarylamine derivatives represent a novel class of tubulin polymerization inhibitors targeting the colchicine binding site and showing significant anti-proliferative activity.

1-(3,4,5-Trimethoxyphenyl)ethane-1,2-diyl esters, a novel compound class with potent chemoreversal activity.

1-(3,4,5-Trimethoxyphenyl)ethane-1,2-diyl esters, which share a fragment from (±)-3'-O-4'-O-bis(3,4-dimethoxycinnamoyl)-cis-khellactone (DMDCK) and 3'R,4'R-disubstituted-2',2'-dimethyldihydropyrano[2,3-f]chromone (DSP), exhibited remarkable chemoreversal activity on multidrug resistant human nasopharyngeal carcinoma (KB) when combined with three anticancer drugs, paclitaxel, vincristine and doxorubicin. Among 15 novel synthesized analogs, bis-trimethoxybenzoyl derivative 15 was the most active (340-fold more active than verapamil when used with vincristine) followed by two di-cinnamoyl derivatives, 10 and 11, and then di-cyclohexanecarbonyl derivative 9. All aliphatic chain derivatives, 3-5, showed no activity. Structure-activity relationship study indicated that a di-ester structure was critical to enhance the activity resulting from the maintenance of the spatial arrangement proposed by the pharmacophore based on the verapamil-binding site. Further mechanism of action study showed 15 inhibited mainly P-glycoprotein efflux pump function, while 13 exhibited an additional multidrug resistance-associated protein efflux pump function.

Design and synthesis of diarylamines and diarylethers as cytotoxic antitumor agents.

Based on a shared structural core of diarylamine in several known anticancer drugs as well as a new cytotoxic hit 6-chloro-2-(4-cyanophenyl)amino-3-nitropyridine (7), 30 diarylamines and diarylethers were designed, synthesized, and evaluated for cytotoxic activity against A549, KB, KB-vin, and DU145 human tumor cell lines (HTCL). Four new leads 11e, 12, 13a, and 13b were discovered with GI(50) values ranging from 0.33 to 3.45µM. Preliminary SAR results revealed that a diarylamine or diarylether could serve as an active structural core, meta-chloro and ortho-nitro groups on the A-ring (either pyridine or phenyl ring) were necessary and crucial for cytotoxic activity, and the para-substituents on the other phenyl ring (B-ring) were related to inhibitory selectivity for different tumor cells. In an investigation of potential biological targets of the new leads, high thoughput kinase screening discovered that new leads 11e, 12 and 13b especially inhibit Mer tyrosine kinase, a proto-oncogene associated with munerous tumor types, with IC(50) values of 2.2-3.0µM. Therefore, these findings provide a good starting point to optimize a new class of compounds as potential anticancer agents, particularly targeting Mer tyrosine kinase.

Antitumor agents 295. E-ring hydroxylated antofine and cryptopleurine analogues as antiproliferative agents: design, synthesis, and mechanistic studies.

Various E-ring hydroxylated antofine and cryptopleurine analogues were designed, synthesized, and tested against five human cancer cell lines. Interesting structure-activity relationship (SAR) correlations were found among these new compounds. The most potent compound 13b was further tested against a series of nonsmall cell lung cancer (NSCLC) cell lines in which it showed impressive antiproliferative activity. Mechanistic studies revealed that 13b is able to down-regulate HSP90 and ß-catenin in A549 lung adenocarcinoma cells in a dose-dependent manner, suggesting a potential use for treating hedgehog pathway-driven tumorigenesis.

Synthesis and evaluation of novel podophyllotoxin analogs.

Because prior studies have shown inconsistency between structure-activity relationships for podophyllotoxin derivatives as topoisomerase II inhibitors and cytotoxic agents, eight novel podophyllotoxin analogs were synthesized to further explore the effects of structural variations on both A and D rings on activity. The new compounds contain a 4,5-dimethoxy substituted A ring and opened D-ring variants and were prepared by appropriate functional and stereochemical operations at the methylenedioxy group, C7, C8, and C8'. Four compounds (15, 18, 21 and 22) demonstrated noticeable inhibitory activity against A549, DU145, KB and KBvin tumor cells, and the most active compound 18 showed IC(50) values less than 10 µg/mL.