Feedback amplification of senolysis using caspase-3-cleavable peptide-doxorubicin conjugate and 2DG.

Therapy-induced senescence (TIS), a common outcome of current cancer therapy, is a known cause of late recurrence and metastasis and thus its eradication is crucial for therapy success. In this study, we introduced a conceptually novel strategy combining radiation-induced apoptosis-targeted chemotherapy (RIATC) with an effective glycolysis inhibitor, 2-deoxy-d-glucose (2DG) to target TIS. RIATC releases cytotoxic payload by amplification, continually increasing TIS, and this can be targeted by 2DG that stimulates an intrinsic apoptotic pathway in senescent cells, the senolysis; the senolytic 2DG also sensitizes cancer cells to chemo/radiation treatment. Anti-tumor efficacy of RIATC was investigated in numerous tumor models, and various cancer types were screened for TIS. Furthermore, in vitro evaluations of molecular markers of senescence, such as senescence-associated ß-galactosidase (SA-ß-Gal) assay, were performed to confirm that TIS was induced by RIATC therapy in MCF-7 cells. The combination therapy with 2DG proved to be effective in MCF-7 tumor-bearing mice that demonstrated feedback amplification of senolysis and successful inhibition of tumor growth. Our findings suggest that RIATC, when given together with 2DG, can overcome therapy-induced senescence and this combination is a promising strategy that enhances the therapeutic benefit of anti-cancer cytotoxic therapy.

Albumin metabolism targeted peptide-drug conjugate strategy for targeting pan-KRAS mutant cancer.

Despite recent breakthroughs in the development of direct KRAS inhibitors and modulators, no drugs targeting pan-KRAS mutant cancers are clinically available. Here, we report a novel strategy to treat pan-KRAS cancers using a caspase-3 cleavable peptide-drug conjugate that exploits enhanced albumin metabolism in KRAS altered cancers to deliver a cytotoxic agent that can induce a widespread bystander killing effect in tumor cells. Increased albumin metabolism in KRAS mutant cancer cells induced apoptosis via the intracellular uptake of albumin-bound MPD1. This allowed caspase-3 upregulation activated MPD1 to release the payload and exert the non-selective killing of neighboring cancer cells. MPD1 exhibited potent and durable antitumor efficacy in mouse xenograft models with different KRAS genotypes. An augmentation of anti-cancer efficacy was achieved by the bystander killing effect derived from the caspase-3 mediated activation of MPD1. In summary, albumin metabolism-induced apoptosis, together with the bystander killing effect of MPD1 boosted by caspase-3 mediated activation, intensified the efficacy of MPD1 in KRAS mutant cancers. These findings suggest that this novel peptide-drug conjugate could be a promising breakthrough for the treatment in the targeting of pan-KRAS mutant cancers.

p53/BNIP3-dependent mitophagy limits glycolytic shift in radioresistant cancer.

The role of p53 in genotoxic therapy-induced metabolic shift in cancers is not yet known. In this study, we investigated the role of p53 in the glycolytic shift in head and neck squamous cell carcinoma cell lines following irradiation. Isogenic p53-null radioresistant cancer cells established through cumulative irradiation showed decreased oxygen consumption and increased glycolysis with compromised mitochondria, corresponding with their enhanced sensitivity to drugs that target glycolysis. In contrast, radioresistant cancer cells with wild-type p53 preserved their primary metabolic profile with intact mitophagic processes and maintained their mitochondrial integrity. Moreover, we identified a previously unappreciated link between p53 and mitophagy, which limited the glycolytic shift through the BNIP3-dependent clearance of abnormal mitochondria. Thus, drugs targeting glycolysis could be used as an alternative strategy for overcoming radioresistant cancers, and the p53 status could be used as a biomarker for selecting participants for clinical trials.

Metronomic oral doxorubicin in combination of Chk1 inhibitor MK-8776 for p53-deficient breast cancer treatment.

Metronomic chemotherapy, which is defined as a low-dose and frequent administration of cytotoxic drugs without drug-free breaks, has been recently emerged as an alternative to traditional MTD therapy and has shown therapeutic benefit in breast cancer patients in numbers of clinical studies. Unlike MTD, metronomic chemotherapy acts by multiple mechanisms including antiangiogenic effect and immunomodulation, but the direct cytotoxic effect only playing a minor role due to the lowered dose. In this light, within the limits of p53-deficient breast cancer, we demonstrate the enhanced anticancer effect of metronomic chemotherapy using doxorubicin when combined with Chk1 inhibitor MK-8776 by specifically augmenting the direct cytotoxic effect on cancer cells. Since the oral drug is greatly favored in metronomic chemotherapy due to the frequent and potential long-term administration, we prepared an oral doxorubicin by producing an ionic complex with deoxycholic acid, which showed sufficient bioavailability and anticancer effect when administered orally. MK-8776 selectively enhanced the cytotoxic effect of low-concentration doxorubicin in p53-deficient breast cancer cells by abrogating the Chk1-dependent cell cycle arrest in vitro. Consistently, combining MK-8776 significantly improved the anticancer effect of the daily administered oral doxorubicin in p53-deficient breast cancer xenografts especially in a lower dose of doxorubicin without evident systemic toxicities. Combination therapy of MK-8776 and metronomic oral doxorubicin would be thus promising in the treatment of p53-deficient breast cancer benefited from the augmented direct cytotoxic effect and low risk of toxicities.

Radiotherapy-assisted tumor selective metronomic oral chemotherapy.

Chemotherapy have commonly been used in maximum tolerated dose to completely eradicate the cancer. However, such treatments often failed due to the complex and dynamic nature of cancer. Therefore, it has been suggested that cancer should be treated as a chronic disease, controlling its growth by providing continuous therapeutic pressure for long-term. Such an approach, however, requires a therapy that is non-toxic and orally available with sufficient potency. Herein, we propose a radiotherapy-assisted orally available metronomic apoptosis-targeted chemotherapy, which delivers doxorubicin continuously to the irradiated tumor with high selectivity while causing minimal toxicities to the normal tissues. DEVD-S-DOX/DCK complex is the anticancer prodrug for our strategy that could selectively release doxorubicin in the irradiated tumor tissue with sufficient oral bioavailability. The prodrug was completely inactive by itself, but displayed potent anticancer activity when coupled with radiotherapy. Consequently, the daily oral administration of DEVD-S-DOX/DCK in combination with the low-dose radiotherapy effectively suppressed the growth of tumor in vivo with no significant systemic toxicities despite that the accumulated dose of doxorubicin exceeded 150 mg/kg. Therefore, the our novel therapy using DEVD-S-DOX/DCK complex is considered as an outstanding treatment option for treating cancer for long-term attributed to its oral availability and low-toxicity profile as well as the potent anticancer effect.

Radiotherapy-associated Furin Expression and Tumor Invasiveness in Recurrent Laryngeal Cancer.

BACKGROUND/AIM: Recurrent laryngeal cancer often shows an aggressive phenotype after radiotherapy and does not respond to conventional therapeutic strategies. In this study, we investigated the contribution of furin to cellular invasiveness in radio-resistant laryngeal cancer. MATERIALS AND METHODS: Using previously established AMC-HN-3 and AMC-HN-8 cell lines from laryngeal carcinoma patients, recurrent laryngeal cancer models were generated by cumulative irradiation (AMC-HN-3-70Gy and AMC-HN-8-70Gy). Immunocytochemistry and western blotting were used to determine the epithelial-mesenchymal transition (EMT). Invasion capacity was assessed using an in vitro invasion assay. Zymography was used to assess metalloproteinase-2 (MMP-2) activity. Tumor xenografts were developed to compare growth rate and furin expression in vivo. Furin expression in 35 patients (45 samples) with salvage total laryngectomy after radiation-based treatment was assessed by laryngeal cancer tissue microarray. RESULTS: Both AMC-HN-3-70Gy and AMC-HN-8-70Gy cell lines underwent EMT following radiation. However, AMC-HN-3-70Gy cells showed increased cellular invasiveness, whereas AMC-HN-8-70Gy cells showed no difference. AMC-HN-3-70Gy cells also exhibited elevated furin expression with up-regulated expression of the active form of membrane type 1-matrix metalloproteinase (MT1-MMP)/MMP-2, whereas AMC-HN-8-70Gy cells did not show significant changes. After administration of a furin inhibitor (chloromethyl ketone (CMK)), AMC-HN-3-70Gy cells showed a significant decrease in MT1-MMP/MMP-2 expression and cellular invasiveness. Nine of 22 samples (40.9%) from salvage total laryngectomy and one of 13 pre-radiation samples (7.7%) had high furin expression. Post-radiation, furin expression increased in seven of 10 patients whose pre- and post-radiation samples were available; all-cancer mortality (three patients) was observed in this group. CONCLUSION: Together with EMT, furin activity may serve as an indicator of an aggressive cancer phenotype, suggesting that furin is a potentially useful target for recurrent laryngeal cancer.

Effect of ß-catenin silencing in overcoming radioresistance of head and neck cancer cells by antagonizing the effects of AMPK on Ku70/Ku80.

BACKGROUND: We attempted to elucidate the mechanism of cell death after radiation by studying how ß-catenin silencing controls the radiation sensitivity of radioresistant head and neck cancer cells. METHODS: The most radioresistant cancer cell line (AMC-HN-9) was selected for study. Targeted silencing of ß-catenin was used on siRNAs. Sensitivity to radiation was examined using clonogenic and methylthiazol tetrazolium (MTT) assays. RESULTS: A combination of irradiation plus ß-catenin silencing led to a significant reduction in the inherent radioresistance of AMC-HN-9 cells. Although expression of Ku70/80 was upregulated in AMC-HN-9 cells after irradiation, Ku70/80 was dramatically decreased in a combination of irradiation and ß-catenin silencing. Interestingly, irradiation-induced Ku70/80 was completely prevented by ß-catenin silencing-induced LKB1/AMP-activated protein kinase (LKB1/AMPK) signal. CONCLUSION: The LKB1/AMPK pathway might relay the signal between the Wnt/ß-catenin pathway and the Ku70/Ku80 DNA repair machinery, and play a decisive role in fine-tuning the responses of cancer cells to irradiation. © 2015 Wiley Periodicals, Inc. Head Neck 38: E1909-E1917, 2016.

Induced phenotype targeted therapy: radiation-induced apoptosis-targeted chemotherapy.

BACKGROUND: Tumor heterogeneity and evolutionary complexity may underlie treatment failure in spite of the development of many targeted agents. We suggest a novel strategy termed induced phenotype targeted therapy (IPTT) to simplify complicated targets because of tumor heterogeneity and overcome tumor evolutionary complexity. METHODS: We designed a caspase-3 specific activatable prodrug, DEVD-S-DOX, containing doxorubicin linked to a peptide moiety (DEVD) cleavable by caspase-3 upon apoptosis. To induce apoptosis locally in the tumor, we used a gamma knife, which can irradiate a very small, defined target area. The in vivo antitumor activity of the caspase-3-specific activatable prodrug combined with radiation was investigated in C3H/HeN tumor-bearing mice (n = 5 per group) and analyzed with the Student's t test or Mann-Whitney U test. All statistical tests were two-sided. We confirmed the basic principle using a caspase-sensitive nanoprobe (Apo-NP). RESULTS: A single exposure of radiation was able to induce apoptosis in a small, defined region of the tumor, resulting in expression of caspase-3. Caspase-3 cleaved DEVD and activated the prodrug. The released free DOX further activated DEVD-S-DOX by exerting cytotoxic effects on neighboring tumor or supporting cells, which repetitively induced the expression of caspase-3 and the activation of DEVD-S-DOX. This sequential and repetitive process propagated the induction of apoptosis. This novel therapeutic strategy showed not only high efficacy in inhibiting tumor growth (14-day tumor volume [mm(3)] vs radiation alone: 848.21 ± 143.24 vs 2511.50 ± 441.89, P < .01) but also low toxicity to normal cells and tissues. CONCLUSION: Such a phenotype induction strategy represents a conceptually novel approach to overcome tumor heterogeneity and complexity as well as to substantially improve current conventional chemoradiotherapy with fewer sequelae and side effects.

Autophagy inhibition can overcome radioresistance in breast cancer cells through suppression of TAK1 activation.

BACKGROUND/AIM: Autophagy is frequently activated in radioresistant cancer cells. In the present study, we evaluated the role of autophagy and transforming growth factor-activated kinase 1 (TAK1) in radioresistance. MATERIALS AND METHODS: TAK1 phosphorylation in MDA-MB231 breast cancer cells was evaluated by western blotting. The regulatory effects of the TAK1 inhibitor and autophagy inhibitor were assessed by cell morphology, cell survival and induction of apoptosis. RESULTS: Radiation induced the phosphorylation of TAK1, whereas the inhibition of TAK1 activity enhanced the cytotoxicity of radiation in MDA-MB231 cells. Autophagy inhibitors significantly enhanced radiation-induced apoptosis of MDA-MB231 cells. This augmentation in radiosensitivity seemed to result from the suppression of TAK1 activation. CONCLUSION: Inhibition of autophagy enhanced radiosensitivity through suppression of radiation-induced TAK1 activation, suggesting that the modulation of TAK1-induced autophagy may be a good therapeutic strategy to treat radioresistant breast cancer.

Enhanced neuronal differentiation of pheochromocytoma 12 cells on polydopamine-modified surface.

Since pheochromocytoma 12 (PC12) cells have the ability of neuronal differentiation upon nerve growth factor (NGF) treatment, they are a good model for studying the neuronal differentiation. Establishing a strong adhesion of PC12 cells to the culture substrate may increase neuronal differentiation, and the use of L-3,4-dihydroxyphenylalanine (L-DOPA), which is responsible for the adhesive property of mussel adhesive proteins (MAPs), is a feasible strategy for such strong adhesion. We hypothesized that a polydopamine-modified surface can promote PC12 cell adhesion and subsequent neuronal differentiation. We examined whether polydopamine-modified surface promotes PC12 cell adhesion, and further evaluated the neuronal differentiation of these cells. The polydopamine modification enhanced the cell adhesion and viability, and also promoted the neuronal differentiation of NGF-stimulated PC12 cells, as evidenced by the elongation of neurites and expression of neuronal differentiation markers, by increasing the activation of NGF/Trk-Rho GTPase signal pathway. Our findings will help develop an improved strategy for functionalizing biomaterial substrates for less-adhesive cells including neural cells.

Tumor targeting efficiency of bare nanoparticles does not mean the efficacy of loaded anticancer drugs: importance of radionuclide imaging for optimization of highly selective tumor targeting polymeric nanoparticles with or without drug.

The better understanding of polymeric nanoparticles as a drug delivery carrier is a decisive factor to get more efficient therapeutic response in vivo. Here, we report the non-invasive imaging of bare polymeric nanoparticles and drug-loaded polymeric nanoparticles to evaluate biodistribution in tumor bearing mice. To make nano-sized drug delivery carrier, glycol chitosan was modified with different degrees of hydrophobic N-acetyl histidine (NAcHis-GC-1, -2, and -3). The biodistribution of polymeric nanoparticles and drug was confirmed by using gamma camera with (131)I-labeled NAcHis-GC and (131)I-labeled doxorubicin (DOX) and by using in vivo live animal imaging with near-infrared fluorescence Cy5.5-labeled NAcHis-GC. Among bare nanoparticles, NAcHis-GC3 (7.8% NAcHis content) showed much higher tumor targeting efficiency than NAcHis-GC1 (3.3% NAcHis content) and NAcHis-GC2 (6.8% NAcHis content). In contrast, for drug-loaded nanoparticles, DOX-NAcHis-GC1 displayed two-fold higher tumor targeting property than DOX-NAcHis-GC3. These data imply that the biodistribution and tumor targeting efficiency between bare and drug-loaded nanoparticles may be greatly different. Therapeutic responses for NAcHis-GC nanoparticles after drug loading were also evaluated. In xenograft animal model, we could find out that DOX-NAcHis-GC1 with higher tumor targeting of DOX has more excellent therapeutic effect than DOX-NAcHis-GC3 and free DOX. These results mean that the hydrophobic core stability might be a critical factor for tumor targeting efficiency of nanoparticles. The present study indicates that by using molecular imaging, we can select more appropriate nanoparticles with the highest tumor targeting properties, leading to exerting more excellent therapeutic results in cancer therapy.