Therapeutic inhibition of polo-like kinases in anaplastic thyroid cancer.

Polo-like kinases (PLKs) are potent regulators of cell proliferation and cell survival. Polo-like kinases are potential targets in the treatment of anaplastic thyroid cancer (ATC), a rare but deadly disease. The therapeutic effects of volasertib, a PLK inhibitor, was evaluated for the treatment of ATC either alone or in combination with sorafenib. Volasertib decreased cell viability in three ATC cell lines (8505C, 8305C, and KAT18) in a dose-dependent manner. Volasertib caused ATC cells to accumulate in G2 /M phase, activated caspase-3 activity, and induced apoptosis. Combination therapy using volasertib and sorafenib in ATC cells showed mostly synergistic effects. In vivo studies revealed that combination therapy of volasertib and sorafenib was effective in the treatment of 8505C xenografts. Single-agent volasertib treatment was sufficient to retard 8305C tumor growth. No substantial morbidity was observed in animals that received either single-agent or combination treatment. These preclinical findings suggest that volasertib could be an effective drug in treating ATC.

Co-exposure to low doses of the food contaminants deoxynivalenol and nivalenol has a synergistic inflammatory effect on intestinal explants.

The global incidence of Fusarium head blight and attendant cereal grains multi-contamination by the trichothecene mycotoxins deoxynivalenol (DON) and nivalenol (NIV) are increasing as a possible result of climate change and inadequate agricultural practices. At the molecular level, these mycotoxins bind to the ribosome, activate the mitogen-activated protein kinase and induce a local and systemic inflammation. DON is of public health concern owing to the narrow margin between exposure and tolerable daily intake. The intestinal inflammatory response to DON, NIV and their mixture was analyzed to determine thresholds for their intestinal pro-inflammatory effects and characterize the type and magnitude of their interaction. Fully differentiated three-dimensional porcine jejunal explants were exposed to increasing doses of DON and NIV alone or in combination; the expression levels of IL-1α, IL-1ß, IL-8, IL-17A and IL-22 were measured by RT-PCR. Doses as low as 0.16 µM DON or 0.73 µM NIV significantly increase the intestinal expression levels of the tested inflammation-related genes. These doses are lower than those previously reported for other intestinal toxicity endpoints. The combined pro-inflammatory activity of DON and NIV was synergistic for all the tested genes with combination index value range of 0.23-0.8. Our results indicate that (1) inflammation is a very sensitive endpoint for the intestinal toxicity of the trichothecenes and (2) co-exposure to DON and NIV has a greater inflammatory effect than induced by mycotoxins alone. This synergy should be taken into account considering the frequent co-occurrence of DON and NIV in the diet.

Multifaceted cytoprotection by synthetic polyacetylenes inspired by the ginseng-derived natural product, panaxytriol.

We describe herein the discovery of a series of panaxytriol (PXT)-derived polyacetylene small molecules with promising cytoprotective activity. In mouse xenograft models, we have demonstrated the capacity of our synthetic analogs to mitigate a range of cancer therapeutic agent-induced toxicities, including body weight loss, lethality, neurotoxicity, and hematotoxicity. Our PXT analogs have also been found to reduce radiation-induced body weight loss and lethality in mouse models. Moreover, several PXT analogs appear to exhibit moderate in vivo antiinflammatory activity as well as in vitro immunoenhancing capabilities. These compounds appear to derive their activity through induction of cancer preventive phase 2 enzymes. The studies described herein suggest that coadministration of a PXT-derived agent with cancer chemotherapeutics or radiation therapy may serve to mitigate a range of therapy-associated toxicities.

Therapeutic inhibition of ATR in differentiated thyroid cancer.

Ataxia telangiectasia and Rad3-related protein (ATR) is a critical component of the DNA damage response and a potential target in the treatment of cancers. An ATR inhibitor, BAY 1895344, was evaluated for its use in differentiated thyroid cancer (DTC) therapy. BAY 1895344 inhibited cell viability in four DTC cell lines (TPC1, K1, FTC-133, and FTC-238) in a dose-dependent manner. BAY 1895344 treatment arrested DTC cells in the G2/M phase, increased caspase-3 activity, and caused apoptosis. BAY 1895344 in combination with either sorafenib or lenvatinib showed mainly synergistic effects in four DTC cell lines. The combination of BAY 1895344 with dabrafenib plus trametinib revealed synergistic effects in K1 cells that harbor BRAFV600E. BAY 1895344 monotherapy retarded the growth of K1 and FTC-133 tumors in xenograft models. The combinations of BAY 1895344 plus lenvatinib and BAY 1895344 with dabrafenib plus trametinib were more effective than any single therapy in a K1 xenograft model. No appreciable toxicity appeared in animals treated with either a single therapy or a combination treatment. Our findings provide the rationale for the development of clinical trials of BAY 1895344 in the treatment of DTC.

Convection enhanced delivery of carboplatin in combination with radiotherapy for the treatment of brain tumors.

The purpose of this study was to further evaluate the therapeutic efficacy of convection enhanced delivery (CED) of carboplatin in combination with radiotherapy for treatment of the F98 rat glioma. Tumor cells were implanted stereotactically into the brains of syngeneic Fischer rats, and 13 or 17 d. later carboplatin (20 µg/10 µl) was administered by either CED over 30 min or by Alzet osmotic pumps (0.5 µg/µl/h for 168 h.) beginning at 7 d after tumor implantation. Rats were irradiated with a 15 Gy fractionated dose (5 Gy × 3) of 6 MV photons to the whole brain beginning on the day after drug administration. Other groups of rats received either carboplatin or X-irradiation alone. The tumor carboplatin concentration following CED of 20 µg in 10 µl was 10.4 µg/g, which was equal to that observed following i.v. administration of 100 mg/kg b.w. Rats bearing small tumors, treated with carboplatin and X-irradiation, had a mean survival time (MST) of 83.4 d following CED and 111.8 d following pump delivery with 40% of the latter surviving >180 d (i.e. cured) compared to 55.2 d for CED and 77.2 d. for pump delivery of carboplatin alone and 31.8 d and 24.2 d, respectively, for X-irradiated and untreated controls. There was no microscopic evidence of residual tumor in the brains of all long-term survivors. Not surprisingly, rats with large tumors had much shorter MSTs. Only modest increases in MSTs were observed in animals that received either oral administration or CED of temozolomide plus X-irradiation (23.2 d and 29.3 d) compared to X-irradiation alone. The present survival data, and those previously reported by us, are among the best ever obtained with the F98 glioma model. Initially, they could provide a platform for a Phase I clinical trial to evaluate the safety and potential therapeutic efficacy of CED of carboplatin in patients with recurrent glioblastomas, and ultimately a Phase II trial of carboplatin in combination with radiation therapy.

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.

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.

Therapeutic effect against human xenograft tumors in nude mice by the third generation microtubule stabilizing epothilones.

The epothilones represent a promising class of natural product-based antitumor drug candidates. Although these compounds operate through a microtubule stabilization mechanism similar to that of taxol, the epothilones offer a major potential therapeutic advantage in that they retain their activity against multidrug-resistant cell lines. We have been systematically synthesizing and evaluating synthetic epothilone congeners that are not accessible through modification of the natural product itself. We report herein the results of biological investigations directed at two epothilone congeners: iso-fludelone and iso-dehydelone. Iso-fludelone, in particular, exhibits a number of properties that render it an excellent candidate for preclinical development, including biological stability, excellent solubility in water, and remarkable potency relative to other epothilones. In nude mouse xenograft settings, iso-fludelone was able to achieve therapeutic cures against a number of human cancer cell lines, including mammarian-MX-1, ovarian-SK-OV-3, and the fast-growing, refractory, subcutaneous neuroblastoma-SK-NAS. Strong therapeutic effect was observed against drug-resistant lung-A549/taxol and mammary-MCF-7/Adr xenografts. In addition, iso-fludelone was shown to exhibit a significant therapeutic effect against an intracranially implanted SK-NAS tumor.

Efficacy of adavosertib therapy against anaplastic thyroid cancer.

Wee1 is a kinase that regulates the G2/M progression by the inhibition of CDK1, which is critical for ensuring DNA damage repair before initiation of mitotic entry. Targeting Wee1 may be a potential strategy in the treatment of anaplastic thyroid cancer, a rare but lethal disease. The therapeutic effects of adavosertib, a Wee1 inhibitor for anaplastic thyroid cancer was evaluated in this study. Adavosertib inhibited cell growth in three anaplastic thyroid cancer cell lines in a dose-dependent manner. Cell cycle analysis revealed cells were accumulated in the G2/M phase. Adavosertib induced caspase-3 activity and led to apoptosis. Adavosertib monotherapy showed significant retardation of the growth of two anaplastic thyroid cancer tumor models. The combination of adavosertib with dabrafenib and trametinib revealed strong synergism in vitro and demonstrated robust suppression of tumor growth in vivo in anaplastic thyroid cancer xenograft models with BRAFV600E mutation. The combination of adavosertib with either sorafenib or lenvatinib also demonstrated synergism in vitro and had strong inhibition of tumor growth in vivo in an anaplastic thyroid cancer xenograft model. No appreciable toxicity appeared in mice treated with either a single agent or combination treatment. Our findings suggest adavosertib holds the promise for the treatment of patients with anaplastic thyroid cancer.

Efficacy and Biomarker Analysis of Adavosertib in Differentiated Thyroid Cancer.

Differentiated thyroid cancer (DTC) patients are usually known for their excellent prognoses. However, some patients with DTC develop refractory disease and require novel therapies with different therapeutic mechanisms. Targeting Wee1 with adavosertib has emerged as a novel strategy for cancer therapy. We determined the effects of adavosertib in four DTC cell lines. Adavosertib induces cell growth inhibition in a dose-dependent fashion. Cell cycle analyses revealed that cells were accumulated in the G2/M phase and apoptosis was induced by adavosertib in the four DTC tumor cell lines. The sensitivity of adavosertib correlated with baseline Wee1 expression. In vivo studies showed that adavosertib significantly inhibited the xenograft growth of papillary and follicular thyroid cancer tumor models. Adavosertib therapy, combined with dabrafenib and trametinib, had strong synergism in vitro, and revealed robust tumor growth suppression in vivo in a xenograft model of papillary thyroid cancer harboring mutant BRAFV600E, without appreciable toxicity. Furthermore, combination of adavosertib with lenvatinib was more effective than either agent alone in a xenograft model of follicular thyroid cancer. These results show that adavosertib has the potential in treating DTC.

Potent DNA-directed alkylating agents: Synthesis and biological activity of phenyl N-mustard-quinoline conjugates having a urea or hydrazinecarboxamide linker.

A series of N-mustard-quinoline conjugates bearing a urea or hydrazinecarboxamide linker was synthesized for antitumor evaluation. The in vitro cytotoxicity studies revealed that compounds with hydrazinecarboxamide linkers were generally more cytotoxic than the corresponding urea counterparts in inhibiting human lymphoblastic leukemia and various solid tumor cell growths in culture. The therapeutic efficacy against human tumor xenografts in animal model was studied. It was shown that complete tumor remission in nude mice bearing human breast carcinoma MX-1 xenograft by 17a, i and 18c, d was achieved. In the present study, it was revealed that both linkers are able to lower the chemically reactive N-mustard pharmacophore and thus the newly synthesized conjugates possess a long half-life in rat plasma. Moreover, the new N-mustard derivatives are able to induce DNA cross-linking either by modified comet assay or by alkaline agarose gel shift assay.

90-kDa heat shock protein inhibition abrogates the topoisomerase I poison-induced G2/M checkpoint in p53-null tumor cells by depleting Chk1 and Wee1.

The G(2)/M cell cycle checkpoint is regulated by a multitude of signaling pathways after genotoxic stress. Herein, we report that treatment with the 90-kDa heat shock protein (Hsp90) molecular chaperone inhibitor 17-allylamino-17-demethoxygeldanamycin (17AAG) selectively abrogates the G(2)/M checkpoint induced by 7-ethyl-10-hydroxycamptothecin (SN-38), an active metabolite of irinotecan, in p53-null compared with p53-intact HCT116 colon cancer cells. The basis for this selectivity can be explained in part by the lack of p21 induction in p53-null cells. In accord with published results, we could show that treatment with 17AAG resulted in depletion of Chk1, a known Hsp90 client protein. In addition, we observed a time- and dose-dependent decrease in Wee1 kinase level, a negative regulator of mitosis, after 17AAG treatment in gastrointestinal cancer cells. Depletion of Wee1 protein preceded mitotic entry induced by 17AAG, and this decrease could be partially rescued by cotreatment with a proteasome inhibitor. Coimmunoprecipitation experiments showed that Hsp90 and Wee1 interacted in whole cells, and 17AAG treatment decreased the degradative half-life of Wee1, indicating that Wee1 is another Hsp90 client in mammalian cells. Knockdown of Chk1 and Wee1 by short interfering RNA each resulted in abrogation of the G(2)/M checkpoint induced by SN-38. The combination of SN-38 and 17AAG was shown to be synergistic in p53-null but not in parental HCT116 cells by median effect/combination index analysis. Taken together, 17AAG specifically inhibits the G(2)/M checkpoint in p53-defective cells by down-regulation of two critical checkpoint kinases, Chk1 and Wee1.

Potent antitumor bifunctional DNA alkylating agents, synthesis and biological activities of 3a-aza-cyclopenta[a]indenes.

A series of bifunctional DNA interstrand cross-linking agents, bis(hydroxymethyl)- and bis(carbamates)-8H-3a-azacyclopenta[a]indene-1-yl derivatives were synthesized for antitumor evaluation. The preliminary antitumor studies revealed that these agents exhibited potent cytotoxicity in vitro and antitumor therapeutic efficacy against human tumor xenografts in vivo. Furthermore, these derivatives have little or no cross-resistance to either Taxol or Vinblastine. Remarkably, complete tumor remission in nude mice bearing human breast carcinoma MX-1 xenograft by 13a,b and 14g,h and significant suppression against prostate adenocarcinoma PC3 xenograft by 13b were achieved at the maximum tolerable dose with relatively low toxicity. In addition, these agents induce DNA interstrand cross-linking and substantial G2/M phase arrest in human non-small lung carcinoma H1299 cells. The current studies suggested that these agents are promising candidates for preclinical studies.

Novel DNA-directed alkylating agents: design, synthesis and potent antitumor effect of phenyl N-mustard-9-anilinoacridine conjugates via a carbamate or carbonate linker.

A series of phenyl N-mustard-9-anilinoacridine conjugates via a carbamate or carbonate linker was synthesized for antitumor evaluation. The carbamate or carbonate linker is able to lower the reactivity of the phenyl N-mustard pharmacophore and thus, these conjugates are rather chemically stable. The in vitro studies revealed that these derivatives possessed significant cytotoxicity with IC(50) in sub-micromolar range in inhibiting human lymphoblastic leukemia (CCRF-CEM), breast carcinoma (MX-1), colon carcinoma (HCT-116) and human non-small cell lung cancer (H1299) cell growth in vitro. Compounds 10a, 10b, 10e, 10i, and 15a were selected for evaluating their antitumor activity in nude mice bearing MX-1 and HCT-116 xenografts. Remarkably, total tumor remission was achieved by these agents with only one cycle of treatment. Interestingly, no tumor relapse was found in mice treated with 10a over 129 days. This agent is capable of inducing DNA interstrand cross-linking in human non-small lung cancer H1299 cells in a dose dependent manner by modified comet assay and has a long half-life in rat plasma.

Synergy of a herpes oncolytic virus and paclitaxel for anaplastic thyroid cancer.

PURPOSE: Novel therapeutic regimens are needed to improve the dismal outcomes of patients with anaplastic thyroid cancer (ATC). Oncolytic herpes simplex virus have shown promising activity against human ATC. We studied the application of oncolytic herpes simplex virus (G207 and NV1023) in combination with currently used chemotherapeutic drugs (paclitaxel and doxorubicin) for the treatment of ATC. EXPERIMENTAL DESIGN AND RESULTS: All four agents showed dose-response cytotoxicity in vitro for the human ATC cell lines KAT4 and DRO90-1. G207, combined with paclitaxel, showed synergistic cytotoxicity. Chou-Talalay combination indices ranged from 0.56 to 0.66 for KAT4, and 0.68 to 0.74 for DRO90-1 at higher affected fractions. Paclitaxel did not enhance G207 viral entry and early gene expression or G207 viral replication. Paclitaxel combined with G207 compared with single-agent treatment or controls showed significantly increased microtubule acetylation, mitotic arrest, aberrant chromatid separation, inhibition of metaphase to anaphase progression, and apoptosis. A single i.t. injection of G207 combined with biweekly i.p. paclitaxel injections in athymic nude mice bearing KAT4 flank tumors showed significantly reduced mean tumor volume (74 +/- 38 mm(3)) compared with G207 alone (388 +/- 109 mm(3)), paclitaxel alone (439 +/- 137 mm(3)), and control (520 +/- 160 mm(3)) groups at 16 days. There was no morbidity in vivo attributable to therapy. CONCLUSIONS: Mechanisms of paclitaxel antitumoral activity, including microtubule acetylation, mitotic block, and apoptosis, were enhanced by G207, which also has direct oncolytic effects. Combination of G207 and paclitaxel therapy is synergistic in treating ATC and holds promise for patients with this fatal disease.

Targeting PLKs as a therapeutic approach to well-differentiated thyroid cancer.

Polo-like kinases (PLKs) are pivotal regulators of cell proliferation and cell survival; therefore, PLKs may be potential targets in the treatment of malignancy. The therapeutic effects of volasertib, a PLKs inhibitor for papillary and follicular thyroid cancer (known as well-differentiated thyroid cancer (WDTC)), were evaluated in this study. Volasertib inhibited cell proliferation in two papillary and two follicular thyroid cancer cell lines in a dose-dependent manner. Volasertib treatment reduced cells in the S phase and increased cells in the G2/M phase. Volasertib activated caspase-3 activity and induced apoptosis. Drug combinations of volasertib and sorafenib showed mostly synergism in four well-differentiated thyroid carcinoma cell lines in vitro. Volasertib treatment in vivo retarded the growth of a papillary thyroid tumor model. Furthermore, the combination of volasertib with sorafenib was more effective than a single treatment of either in a follicular thyroid cancer xenograft model. Promising safety profiles appeared in animals treated with either volasertib alone or volasertib and sorafenib combination therapy. These findings support volasertib as a potential drug for the treatment of patients with WDTC.

Synthesis and biological activity of stable and potent antitumor agents, aniline nitrogen mustards linked to 9-anilinoacridines via a urea linkage.

To improve the chemical stability and therapeutic efficacy of N-mustard, a series of phenyl N-mustard linked to DNA-affinic 9-anilinoacridines and acridine via a urea linker were synthesized and evaluated for antitumor studies. The new N-mustard derivatives were prepared by the reaction of 4-bis(2-chloroethyl)aminophenyl isocyanate with a variety of 9-anilinoacridines or 9-aminoacridine. The antitumor studies revealed that these agents exhibited potent cytotoxicity in vitro without cross-resistance to taxol or vinblastine and showed potent antitumor therapeutic efficacy in nude mice against human tumor xenografts. It also showed that 24d was capable of inducing marked dose-dependent levels of DNA cross-linking by comet assay and has long half-life in rat plasma.

Activity of roniciclib in medullary thyroid cancer.

Altered cyclin-dependent kinase activity is observed in many human malignancies. Cyclin-dependent kinases that promote cell cycle progression may be promising targets in the treatment of cancer. The therapeutic effects of roniciclib, a cyclin-dependent kinase inhibitor for medullary thyroid cancer were investigated in the present study. Roniciclib inhibited medullary thyroid cancer cell proliferation in a dose-dependent manner. Roniciclib induced caspase-3 activity and contributed to apoptosis. Cell cycle progression was arrested in the G2 phase. In vivo, roniciclib treatment retarded the growth of tumors of medullary thyroid cancer xenografts. In addition, roniciclib in combination with sorafenib was more effective than either single treatment in a xenograft model. No morbidity was observed in animals treated with single roniciclib therapy and combination treatment of roniciclib and sorafenib. These data provide a rationale for clinical assessment of using roniciclib in the treatment of patients with medullary thyroid cancer.

Potent effects of roniciclib alone and with sorafenib against well-differentiated thyroid cancer.

Activation of cyclin-dependent kinase activity is frequently observed in many human cancers; therefore, cyclin-dependent kinases that promote cell cycle transition and cell proliferation may be potential targets in the treatment of malignancy. The therapeutic effects of roniciclib, a cyclin-dependent kinase inhibitor for papillary and follicular thyroid cancer (designated as well-differentiated thyroid cancer), were investigated in this study. Roniciclib inhibited cell proliferation in two papillary and two follicular thyroid cancer cell lines in a dose-dependent manner. Roniciclib activated caspase-3 activity and induced apoptosis. Cell cycle progression was arrested in the G2/M phase. Roniciclib treatment in vivo retarded the growth of two well-differentiated thyroid tumors in xenograft models in a dose-dependent fashion. Furthermore, the combination of roniciclib with sorafenib was more effective than either single treatment in a follicular thyroid cancer xenograft model. Acceptable safety profiles appeared in animals treated with either roniciclib alone or roniciclib and sorafenib combination therapy. These findings support roniciclib as a potential drug for the treatment of patients with well-differentiated thyroid cancer.