In patients with triple‐negative breast cancer (TNBC), evidence suggests that tumor‐initiating cells (TIC) have stem cell‐like properties, leading to invasion and metastasis. HSP90 plays a critical role in the conformational maintenance of many client proteins in TIC development. Therefore, we hypothesize that the novel C‐terminal HSP90 inhibitors KU711 and KU758 can target TIC and represent a promising strategy for overcoming metastasis. Human breast cancer cells (MDA‐MB‐468LN, MDA‐MB‐231) treated with the HSP90 inhibitors KU711, KU758, and 17‐AAG showed a 50–80% decrease in TIC markers CD44 and aldehyde dehydrogenase (P < 0.01) as assessed by flow cytometry. A decrease in sphere formation, which was used to assess self‐renewal, was observed after the treatment of TNBC cells starting at 2.5 µm KU711 and 0.31 µm KU758. KU compounds also blocked the invasion and migration of TNBC cells in a dose‐dependent manner. The knockdown of HSP90 clients was observed without any change in prosurvival HSP70 levels. In vivo, in a murine orthotopic breast cancer model, treatment with KU758 and KU711 yielded an approximately twofold and a fourfold reduction in tumor volumes versus control, respectively, without demonstrated toxicity. In conclusion, C‐terminal HSP90 inhibitors are potent novel therapeutics against TNBC in vitro and in vivo as they target TICs and block invasion, EMT transition, and self‐renewal.
The novel clopidogrel conjugate, DT‐678, is an effective inhibitor of platelets and thrombosis in preclinical studies. However, a comparison of the bleeding risk with DT‐678 and currently approved P2Y 12 antagonists has yet to be determined. The objective of this study was to evaluate the bleeding tendency of animals treated with clopidogrel, ticagrelor, and DT‐678. Ninety‐one New Zealand white rabbits were randomized to one of 13 treatment groups (n = 7). Platelet activation was assessed by flow cytometry and light transmission aggregometry before and after the administration of various doses of DT‐678, clopidogrel, and ticagrelor. Tongue template bleeding times were also measured before and after drug treatment. Treatment with P2Y 12 receptor antagonists caused a dose‐dependent reduction in markers of platelet activation (P‐selectin and integrin α IIb β 3 ) and aggregation in response to adenosine diphosphate stimulation. At the same doses required for platelet inhibition, clopidogrel and ticagrelor significantly prolonged bleeding times, while DT‐678 did not. DT‐678 and the FDA‐approved P2Y 12 antagonists clopidogrel and ticagrelor are effective inhibitors of platelet activation and aggregation. However, unlike clopidogrel and ticagrelor, DT‐678 did not prolong bleeding times at equally effective antiplatelet doses. The results suggest a more favorable benefit/risk ratio for DT‐678 and potential utility as part of a dual antiplatelet therapy regimen.
Introduction: Adrenocortical carcinoma (ACC) is a rare endocrine malignancy, with very poor prognosis as a majority of the patients have advanced disease at the time of diagnosis. Currently, adjuvant therapy for most patients consists of either mitotane (M) alone or in combination with multi-drug chemotherapeutics such as etoposide (E), doxorubicin (D), and cisplatin (P), known as the Italian protocol (IP; EDPM). This multi-drug treatment regimen, however, carries significant toxicity potential for patients. One way to improve toxicity profiles with these drugs in combination is to understand where their synergy occurs and over what dosing range so that lower dose regimens could be applied in combination with equal or improved efficacy. We hypothesize that a better understanding of the synergistic effects as well as the regulation of steroidogenic enzymes during combination therapy may provide more optimized combinational options with good potency and lower toxicity profiles. Methods: Two human ACC cell lines, NCI-H295R (hormonally active) and SW13 (hormonally inactive), were grown in 2D culture in appropriate growth medium. The viability of the cells after treatment with varying concentrations of the drugs (E, D, and P) either alone or in combinations with M was determined using the CellTiter Glow assay after 72 h, and the combination index for each was calculated using Compusyn by the Chou–Talalay method. The expression levels of enzymes associated with steroidogenesis were evaluated by RT-PCR in NCI-H295R. Results: When both cell lines were treated with M (ranging 25–50 μM), +E (ranging 18.75–75 μM), and +D (ranging 0.625–2.5 μM) we observed a synergistic effect (CI < 1) with potency equivalent to the full Italian protocol (IP), whereas combining M + P + D had an antagonistic effect (CI > 1) indicating the negative effect of adding cisplatin in the combination. Comparing the hormonally active and inactive cell lines, M + P + E was antagonistic in NCI-H295R and synergistic in SW13. Treatment of NCI-H295R cells with antagonistic combinations (M + P + D, M + P + E) resulted in a significant decrease in the levels of steroidogenic enzymes STAR, CYP11A1, and CYP21A2 compared to IP (p < 0.05) while M + E + D resulted in increased expression or no significant effect compared to IP across all genes tested. Conclusions: The synergistic effect for M + E + D was significant and equivalent in potency to the full IP in both cell lines and resulted in a steroidogenic gene expression profile similar to or better than that of full IP, warranting further evaluation. Future in vivo evaluation of the combination of M + E + D (with removal of P from the IP regimen) may lower toxicity while maintaining anticancer efficacy in ACC.
Arterial thrombosis is the leading cause of death in the developed world and hypertensive patients are at an increased risk. Dual antiplatelet therapy, consisting of low-dose aspirin in combination with a P2Y 12 antagonist, is the most common preventative treatment for arterial thrombosis. Clopidogrel, a P2Y 12 antagonist, is associated with cerebral microbleeds and intracerebral hemorrhages. We have previously reported in rabbits that clopidogrel (3mg/kg) induces bleeding at lower doses than required to inhibit platelet aggregation, making it unlikely that the clopidogrel-mediated bleeding events are the result of platelet inhibition. I hypothesized that clopidogrel was acting on other purinergic receptors to impair artery function due to the structural similarity of the metabolites of clopidogrel. Purinergic receptors, P2Y 1 , P2Y 2 , P2Y 4 and P2Y 6 , are expressed in the endothelium of cerebral vessels. The purpose of this study was to determine the effects of clopidogrel on vascular function in response to activation of P2Y 1 , P2Y 2 , P2Y 4 and P2Y 6 . The middle cerebral artery (MCA) was used as it is the site most common for cerebrovascular events. New Zealand white rabbits (n=3) were treated for 3 days with vehicle or clopidogrel (3mg/kg/day) prior to MCA isolation. Myogenic tone was allowed to develop before assessing the response of the MCA to increasing concentrations (10 -9 -10 -5 M) of specific purinergic receptor agonists P2Y 1 : MRS 2365; P2Y 2 : MRS 2768; P2Y 4 : MRS 4062; P2Y 6 : MRS 2693. If myogenic tone was not generated the MCA was preconstricted with 10 -6 M serotonin. Response was analyzed by two-way ANOVA and compared to vehicle using Dunnett’s post-test. In all cases, p < 0.05 denote statistical significance. Constriction was calculated as a percent change from baseline. Stimulation with the P2Y 4 agonist resulted in a mean maximal constriction of 28.95% (28.95 of 100) for vehicle and 52.72% (52.72 of 100) for 3mg/kg clopidogrel. There was no significant difference upon stimulation with the P2Y 1 , P2Y 2 , or P2Y 6 agonists compared to vehicle. These data suggest that clopidogrel has vascular effects on the P2Y 4 receptor expressed in the MCA and may explain the cerebral bleeding side effects observed.
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