Low-Dose Rifabutin Increases Cytotoxicity in Antimitotic-Drug-Treated Resistant Cancer Cells by Exhibiting Strong P-gp-Inhibitory Activity.

The cytotoxicity of various antibiotics at low doses in drug-resistant cancer cells was evaluated. Low doses of rifabutin were found to markedly increase the cytotoxicity of various antimitotic drugs, such as vincristine (VIC), to P-glycoprotein (P-gp)-overexpressing antimitotic-drug-resistant KBV20C cells. Rifabutin was also found to exert high levels of P-gp-inhibitory activity at 4 and 24 h posttreatment, suggesting that the cytotoxicity of VIC + rifabutin was mainly due to the direct binding of rifabutin to P-gp and the reduction of VIC efflux by P-gp. The combination of VIC + rifabutin also increased early apoptosis, G2 arrest, and the DNA damaging marker, pH2AX protein. Interestingly, only the combination of VIC + rifabutin induced remarkable levels of cytotoxicity in resistant KBV20C cells, whereas other combinations (VIC + rifampin, VIC + rifapentine, and VIC + rifaximin) induced less cytotoxicity. Such finding suggests that rifabutin specifically increases the cytotoxicity of VIC in KBV20C cells, independent of the toxic effect of the ansamycin antibiotic. Only rifabutin had high P-gp-inhibitory activity, which suggests that its high P-gp-inhibitory activity led to the increased cytotoxicity of VIC + rifabutin. As rifabutin has long been used in the clinic, repositioning this drug for P-gp-overexpressing resistant cancer could increase the availability of treatments for patients with drug-resistant cancer.

JAK2 Inhibitor, Fedratinib, Inhibits P-gp Activity and Co-Treatment Induces Cytotoxicity in Antimitotic Drug-Treated P-gp Overexpressing Resistant KBV20C Cancer Cells.

P-glycoprotein (P-gp) overexpression is one of the major mechanisms of multidrug resistance (MDR). Previously, co-treatment with Janus kinase 2 (JAK2) inhibitors sensitized P-gp-overexpressing drug-resistant cancer cells. In this study, we assessed the cytotoxic effects of JAK2 inhibitor, fedratinib, on drug-resistant KBV20C cancer cells. We found that co-treatment with fedratinib at low doses induced cytotoxicity in KBV20C cells treated with vincristine (VIC). However, fedratinib-induced cytotoxicity was little effect on VIC-treated sensitive KB parent cells, suggesting that these effects are specific to resistant cancer cells. Fluorescence-activated cell sorting (FACS), Western blotting, and annexin V analyses were used to further investigate fedratinib's mechanism of action in VIC-treated KBV20C cells. We found that fedratinib reduced cell viability, increased G2 arrest, and upregulated apoptosis when used as a co-treatment with VIC. G2 phase arrest and apoptosis in VIC-fedratinib-co-treated cells resulted from the upregulation of p21 and the DNA damaging marker pH2AX. Compared with dimethyl sulfoxide (DMSO)-treated cells, fedratinib-treated KBV20C cells showed two-fold higher P-gp-inhibitory activity, indicating that VIC-fedratinib sensitization is dependent on the activity of fedratinib. Similar to VIC, fedratinib co-treatment with other antimitotic drugs (i.e., eribulin, vinorelbine, and vinblastine) showed increased cytotoxicity in KBV20C cells. Furthermore, VIC-fedratinib had similar cytotoxic effects to co-treatment with other JAK2 inhibitors (i.e., VIC-CEP-33779 or VIC-NVP-BSK805) at the same dose; similar cytotoxic mechanisms (i.e., early apoptosis) were observed between treatments, suggesting that co-treatment with JAK2 inhibitors is generally cytotoxic to P-gp-overexpressing resistant cancer cells. Given that fedratinib is FDA-approved, our findings support its application in the co-treatment of P-gp-overexpressing cancer patients showing MDR.

Terconazole, an Azole Antifungal Drug, Increases Cytotoxicity in Antimitotic Drug-Treated Resistant Cancer Cells with Substrate-Specific P-gp Inhibitory Activity.

Azole antifungal drugs have been shown to enhance the cytotoxicity of antimitotic drugs in P-glycoprotein (P-gp)-overexpressing-resistant cancer cells. Herein, we examined two azole antifungal drugs, terconazole (TCZ) and butoconazole (BTZ), previously unexplored in resistant cancers. We found that both TCZ and BTZ increased cytotoxicity in vincristine (VIC)-treated P-gp-overexpressing drug-resistant KBV20C cancer cells. Following detailed analysis, low-dose VIC + TCZ exerted higher cytotoxicity than co-treatment with VIC + BTZ. Furthermore, we found that VIC + TCZ could increase apoptosis and induce G2 arrest. Additionally, low-dose TCZ could be combined with various antimitotic drugs to increase their cytotoxicity in P-gp-overexpressing antimitotic drug-resistant cancer cells. Moreover, TCZ exhibited P-gp inhibitory activity, suggesting that the inhibitory activity of P-gp plays a role in sensitization afforded by VIC + TCZ co-treatment. We also evaluated the cytotoxicity of 12 azole antifungal drugs at low doses in drug-resistant cancer cells. VIC + TCZ, VIC + itraconazole, and VIC + posaconazole exhibited the strongest cytotoxicity in P-gp-overexpressing KBV20C and MCF-7/ADR-resistant cancer cells. These drugs exerted robust P-gp inhibitory activity, accompanied by calcein-AM substrate efflux. Given that azole antifungal drugs have long been used in clinics, our results, which reposition azole antifungal drugs for treating P-gp-overexpressing-resistant cancer, could be employed to treat patients with drug-resistant cancer rapidly.

Acute toxicity, oxidative stress, and apoptosis due to short-term triclosan exposure and multi- and transgenerational effects on in vivo endpoints, antioxidant defense, and DNA damage response in the freshwater water flea Daphnia magna.

In this study, we measured the acute toxicity of triclosan (TCS) in neonate and adult Daphnia magna water fleas. The median lethal concentrations were 184.689 and 349.511 µg/L, respectively. Oxidative stress induced by TCS was analyzed based on changes in reactive oxygen species (ROS) content and antioxidant enzymatic activities in D. magna. Based on these endpoints, TCS concentrations of 50 and 100 µg/L induced oxidative stress. However, several apoptosis-mediated proteins showed TCS-induced oxidative-stress damage in response to 25 µg/L, indicating that apoptotic proteins were the most sensitive mediators. We also evaluated the multi- and transgenerational effects of TCS on D. magna over three generations in terms of various in vivo endpoints, DNA damage responses, and biochemical reactions. The transgenerational group exposed to TCS exhibited greater negative impacts on antioxidant responses, DNA fragmentation status, and biological endpoints compared with the multigenerational exposure group, leading to decreased reproductive rates and higher ROS content. The transcriptional expression levels of glutathione S-transferase genes in the transgenerational exposure group were upregulated compared to those in the multigenerational group but were fully recovered in F2 offspring. Our findings provide an in-depth understanding of the adaptive effects of multigenerational exposure to TCS.

Synergistic toxic mechanisms of microplastics and triclosan via multixenobiotic resistance (MXR) inhibition-mediated autophagy in the freshwater water flea Daphnia magna.

Since a mixed state of environmental contaminants, including microplastics (MPs), heavy metals, pharmaceuticals, and personal care products (PPCPs), exists in aquatic ecosystems, it is necessary to evaluate not only the adverse effects of exposure to a single stressor but to combined stressors. In this study, we exposed the freshwater water flea Daphnia magna to 2 µm MPs and triclosan (TCS), one of PPCPs, for 48 h to investigate the synergistic toxic consequences of simultaneous exposure to both pollutants. We measured in vivo endpoints, antioxidant responses, multixenobiotic resistance (MXR) activity, and autophagy-related protein expression via the PI3K/Akt/mTOR and MAPK signaling pathways. While MPs single exposure did not show toxic effects in water fleas, simultaneous exposure to TCS and MPs was associated with significantly greater deleterious effects in the form of increased mortality and alterations in antioxidant enzymatic activities compared with water fleas exposed to TCS alone. In addition, MXR inhibition was confirmed by measurement of the expression of P-glycoproteins and multidrug-resistance proteins in MPs-exposed groups, which led to the accumulation of TCS. Overall, these results suggest that simultaneous exposure to MPs and TCS resulted in higher TCS accumulation via MXR inhibition, leading to synergistic toxic effects such as autophagy in D. magna.

Co-treatment of Low Dose Pacritinib, a Phase III Jak2 Inhibitor, Greatly Increases Apoptosis of P-gp Over-expressing Cancer Cells With Multidrug Resistance.

BACKGROUND/AIM: The over-expression of P-glycoprotein (P-gp) is a major mechanism underlying multidrug resistance (MDR). Co-treatment with Janus kinase 2 (Jak2) inhibitors sensitizes P-gp-over-expressing drug-resistant cancer cells. In this study, we evaluated pacritinib, a Jak2 inhibitor currently in phase III clinical trials. MATERIALS AND METHODS: Microscopic observation, cell viability assay, colony forming assay, rhodamine uptake tests, annexin V analyses, fluorescence-activated cell sorting (FACS), and western-blot analysis were performed to further investigate the mechanism of action. RESULTS: We found that pacritinib reduced cell viability, induced G2 arrest, and upregulated early apoptosis when administered to P-gp-over-expressing resistant KBV20C cells with vincristine (VIC). Moreover, apoptosis and G2 arrest in VIC-pacritinib-treated cells were involved in the upregulation of pH2AX expression. Pacritinib had an approximately 2-fold higher P-gp-inhibitory activity than the dimethyl sulfoxide (DMSO)-treated control, indicating that VIC-pacritinib sensitization involves the P-gp-inhibitory effects of pacritinib. Similar to VIC, other antimitotic drugs (vinorelbine, vinblastine, and eribulin) could also sensitize against KBV20C cells by co-treatment with pacritinib. Furthermore, comparison of pacritinib with previously characterized Jak2 inhibitors revealed that the VIC-pacritinib combination had sensitization effects similar to those of VIC- CEP-33779 or VIC-NVP-BSK805 combinations at lower doses in KBV20C cells. Generally, Jak2 inhibitor and VIC co-treatment sensitized P-gp-over-expressing resistant cancer cells by inducing early apoptosis. CONCLUSION: Collectively, pacritinib, induced G2 arrest, reduced cell viability, had high P-gp inhibitory activity, and upregulated the expression of pH2AX when used in combination with VIC. As pacritinib is a Jak2 inhibitor currently in phase III clinical trials, our findings may facilitate the application of this co-treatment in patients with MDR cancer.

Combination Treatment Using Pyruvate Kinase M2 Inhibitors for the Sensitization of High Density Triple-negative Breast Cancer Cells.

BACKGROUND/AIM: Few studies have examined the correlation between pyruvate kinase M2 (PKM2) overexpression and triple-negative breast cancer (TNBC). TNBC is considered incurable with the currently available treatments, highlighting the need for alternative therapeutic targets. MATERIALS AND METHODS: PKM2 expression was examined immunohistochemically in human breast tumor samples. Furthermore, we studied the effect of three PKM2 inhibitors (gliotoxin, shikonin, and compound 3K) in the MDA-MB-231 TNBC cell line. RESULTS: PKM2 overexpression correlates with TNBC. Interestingly, most TNBC tissues showed increased levels of PKM2 compared to those of receptor-positive breast cancer tissues. This suggests that PKM2 overexpression is an important factor in the development of TNBC. MDA-MB-231 TNBC cells are resistant to anticancer drugs, such as vincristine (VIC) compared to other cancer cells. We found that the recently developed PKM2 inhibitor gliotoxin sensitized MDA-MB-231 cells at a relatively low dose to the same extent as the known PKM2 inhibitor shikonin, suggesting that PKM2 inhibitors could be an effective treatment for TNBC. Detailed sensitization mechanisms were also analyzed. Both gliotoxin and shikonin highly increased late apoptosis in MDA-MB-231 cells, as revealed by annexin V staining. However, MDA-MB-231 cells with high cellular density inhibited the sensitizing effect of PKM2 inhibitors; therefore, we investigated ways to overcome this inhibitory effect. We found that gliotoxin+shikonin co-treatment highly increased toxicity in MDA-MB-231 cells with high density, whereas either VIC+gliotoxin or VIC+shikonin were not effective. Thus, combination therapy with various PKM2 inhibitors may be more effective than combination therapy with anticancer drugs. Gliotoxin+shikonin co-treatment did not increase S or G2 arrest in cells, suggesting that the co-treatment showed a high increase in apoptosis without S or G2 arrest. We confirmed that another recently developed PKM2 inhibitor compound 3K had similar mechanisms of sensitizing MDA-MB-231 cells, suggesting that PKM2 inhibitors have similar sensitization mechanisms in TNBC. CONCLUSION: PKM2 is a regulator of the oncogenic function of TNBC, and combination therapy with various PKM2 inhibitors may be effective for high-density TNBC. Targeting PKM2 in TNBC lays the foundation for the development of PKM2 inhibitors as promising anti-TNBC agents.

PKM2 Is Overexpressed in Glioma Tissues, and Its Inhibition Highly Increases Late Apoptosis in U87MG Cells With Low-density Specificity.

BACKGROUND/AIM: Pyruvate kinase M2 (PKM2) functions as an important rate-limiting enzyme in aerobic glycolysis and is involved in tumor initiation and progression. However, there are few studies on the correlation between PKM2 expression and its role in glioma. MATERIALS AND METHODS: PKM2 expression was immunohistochemically examined in human brain tumor samples. Furthermore, we studied the effects of two PKM2 inhibitors (shikonin and compound 3K) on the U87MG glioma cell line. RESULTS: PKM2 was overexpressed in most glioma tissues when compared to controls. Interestingly, glioma-adjacent tissues from showed slight PKM2 overexpression. This suggests that PKM2 overexpression maybe an important trigger factor for glioma tumorigenesis. We found that the PKM2 inhibitor shikonin was effective against U87MG cells at a relatively low dose and was largely dependent on low cellular density compared to the effects of the anticancer drug vincristine. Shikonin highly increased late-apoptosis of U87MG cells. We also demonstrated that autophagy was involved in the increase in late-apoptosis levels caused by shikonin. Although vincristine treatment led to a high level of G2-phase arrest in U87MG cells, shikonin did not increase G2 arrest. Co-treatment with two PKM2 inhibitors, shikonin and compound 3K, increased the inhibitory effects. CONCLUSION: Combination therapy with PKM2 inhibitors together might be more effective than combination therapy with anticancer drugs. Our findings encourage the application of PKM2-targeting in gliomas, and lay the foundation for the development of PKM2 inhibitors as promising antitumor agents for glioma.

Sensitization Effects of Repurposed Blood Pressure-regulating Drugs on Drug-resistant Cancer Cells.

BACKGROUND/AIM: We investigated drugs that could sensitize KBV20C cancer cells resistant to eribulin or vincristine (VIC) treatment and assessed their associated mechanisms of action. MATERIALS AND METHODS: Such cancer cells were known to overexpress P-glycoprotein (P-gp). Considering that reserpine (P-gp inhibitor) plays a regulatory role in patients with high blood pressure, we investigated the effect of low doses of 27 blood pressure-regulating drugs on VIC-resistant KBV20C cells. This was done to identify drugs that could be repurposed for sensitizing antimitotic drug-resistant KBV20C cells at relatively low doses. Fluorescence-activated cell sorting (FACS), annexin V analyses, rhodamine uptake tests and western-blot analysis were performed to further investigate the mechanism of action of such drugs. RESULTS: We found that co-treatment with amiodarone, nicardipine, carvedilol, or vardenafil at low doses could highly sensitize KBV20C cells treated with eribulin or VIC. These drugs reduced cellular viability, increased G2 arrest and up-regulated apoptosis when co-administered with eribulin or VIC. Considering that they sensitize with either co-treatment of eribulin or VIC, we assumed that they can be combined with other antimitotic drugs to sensitize the resistant cancer cells. Through detailed quantitative analysis, we found that eribulin with amiodarone had a higher sensitization effect than eribulin with nicardipine or eribulin with carvedilol. We found that reserpine had the highest P-gp-inhibitory activity, indicating that eribulin- or VIC-reserpine sensitization involves the P-gp inhibitory effects of reserpine. However, we found that amiodarone, nicardipine, carvedilol and vardenafil had very low P-gp inhibitory activity. Moreover, we found that cells co-treated with VIC-carvedilol down-regulated expression of pERK. CONCLUSION: Highly antimitotic drug-resistant KBV20C cells can be sensitized by co-treatment with the repurposed blood pressure-regulating drugs amiodarone, nicardipine, carvedilol or vardenafil. These findings indicate that the repurposed blood pressure-regulating drugs may potentially be used in drug-resistant cancer patients without any toxic effects due to P-gp inhibition.