Senolysis of gemcitabine-induced senescent human pancreatic cancer cells.

INTRODUCTION: Gemcitabine (GEM) is often used to treat pancreatic cancer. Many anti-cancer drugs induce cancer cell death, but some cells survive after cell cycle arrest. Such a response to DNA damage is termed cellular senescence. Certain drugs, including the Bcl-2-family inhibitor ABT-263, kill senescent cells; this is termed senolysis. In this study, we examined the therapeutic benefits of ABT-263 in GEM-induced senescence of human pancreatic cancer cells. METHODS AND RESULTS: Of four pancreatic cancer cell lines (PANC-1, AsPC-1, CFPAC-1, and PANC10.05), GEM induced senescent features in PANC-1 and AsPC-1 cells, including increases in the cell sizes and expression levels of mRNAs encoding interleukin (IL)-6/IL-8 and induction of ß-galactosidase. Successive treatment with GEM and ABT-263 triggered apoptosis in PANC-1 and AsPC-1 cells and suppressed colony formation significantly. Senolysis of GEM-induced senescent pancreatic cancer cells by ABT-263 was triggered by a Bcl-xL inhibitor, but not by a Bcl-2 inhibitor, suggesting a central role for Bcl-xL in senolysis. In a xenograft mouse model, combined treatment with GEM and ABT-737 (an ABT-263 analog exhibiting the same specificity) suppressed in vivo growth of AsPC-1 significantly. CONCLUSION: Together, our results indicate that sequential treatment with GEM and senolytic drugs effectively kill human pancreatic cancer cells.

Hypoxia-related carbonic anhydrase 9 induces serpinB9 expression in cancer cells and apoptosis in T cells via acidosis.

Hypoxia is a common feature of solid tumors. However, the impact of hypoxia on immune cells within tumor environments remains underexplored. Carbonic anhydrase 9 (CA9) is a hypoxia-responsive tumor-associated enzyme. We previously noted that regardless of human CA9 (hCA9) expression, hCA9-expressing mouse renal cell carcinoma RENCA (RENCA/hCA9) presented as a "cold" tumor in syngeneic aged mice. This study delves into the mechanisms behind this observation. Gene microarray analyses showed that RENCA/hCA9 cells exhibited elevated mouse serpinB9, an inhibitor of granzyme B, relative to RENCA cells. Corroborating this, RENCA/hCA9 cells displayed heightened resistance to antigen-specific cytotoxic T cells compared with RENCA cells. Notably, siRNA-mediated serpinB9 knockdown reclaimed this sensitivity. In vivo tests showed that serpinB9 inhibitor administration slowed RENCA tumor growth, but this effect was reduced in RENCA/hCA9 tumors, even with adjunctive immune checkpoint blockade therapy. Further, inducing hypoxia or introducing the mouse CA9 gene upregulated serpinB9 expression, and siRNA-mediated knockdown of the mouse CA9 gene inhibited the hypoxia-induced induction of serpinB9 in the original RENCA cells. Supernatants from RENCA/hCA9 cultures had lower pH than those from RENCA, suggesting acidosis. This acidity enhanced serpinB9 expression and T cell apoptosis. Moreover, coculturing with RENCA/hCA9 cells more actively prompted T cell apoptosis than with RENCA cells. Collectively, these findings suggest hypoxia-associated CA9 not only boosts serpinB9 in cancer cells but also synergistically intensifies T cell apoptosis via acidosis, characterizing RENCA/hCA9 tumors as "cold."

A modulatory effect of L-arginine supplementation on anticancer effects of chemoimmunotherapy in colon cancer-bearing aged mice.

Myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) are increased in cancer-bearing aged hosts. Arginase-I in MDSCs degrades L-arginine, an amino acid required for T cell activation and proliferation. In this study, we compared the therapeutic efficacy of 5-fluorouracil (5-FU)/oxaliplatin (L-OHP) and cyclophosphamide (CP) between young and aged colon cancer-bearing mice. Therapy with 5-FU/L-OHP and CP significantly suppressed the in vivo growth of CT26 and MC38 colon carcinomas in syngeneic young mice, whereas this effect was attenuated in aged mice. L-arginine monotherapy showed no effect in aged mice. However, additional therapy with anti-programmed cell death (PD)-1 antibody and L-arginine supplementation boosted the effect of chemoimmunotherapy in aged mice, and some mice were cured. During all combination therapy, tumor-specific cytotoxic T lymphocytes (CTLs) were generated from mice with non-progressing tumor, but not from those with progressing tumor. Plasma L-arginine levels were lower in aged than young mice, and chemotherapy tended to decrease the plasma L-arginine levels in aged mice. Compared to young mice, CT26-bearing aged mice decreased arginase activity, arginase-I expression, and the proportion of monocytic MDSCs in tumor tissues, whereas contrasting results were observed in MC38-bearing aged mice. Importantly, the induction of tumor-specific CTLs was impaired at lower doses of L-arginine in vitro, and the infiltration of CTLs into CT26 tissues after chemoimmunotherapy was promoted by L-arginine administration in vivo. These results indicate that chemoimmunotherapy was less effective in cancer-bearing aged mice, but that L-arginine supplementation can modulate its therapeutic efficacy via its effect on tumor-specific CTLs.

Humanized liver TK-NOG mice with functional deletion of hepatic murine cytochrome P450s as a model for studying human drug metabolism.

Chimeric TK-NOG mice with a humanized liver (normal Hu-liver) are a unique animal model for predicting drug metabolism in humans. However, residual mouse hepatocytes occasionally prevent the precise evaluation of human drug metabolism. Herein, we developed a novel humanized liver TK-NOG mouse with a conditional knockout of liver-specific cytochrome P450 oxidoreductase (POR cKO Hu-liver). Immunohistochemical analysis revealed only a few POR-expressing cells around the portal vein in POR cKO mouse livers. NADPH-cytochrome c reductase and cytochrome P450 (P450)-mediated drug oxidation activity in liver microsomes from POR cKO mice was negligible. After the intravenous administration of S-warfarin, high circulating and urinary levels of S-7-hydroxywarfarin (a major human metabolite) were observed in POR cKO Hu-liver mice. Notably, the circulating and urinary levels of S-4'-hydroxywarfarin (a major warfarin metabolite in mice) were much lower in POR cKO Hu-liver mice than in normal Hu-liver mice. POR cKO Hu-liver mice with minimal interference from mouse hepatic P450 oxidation activity are a valuable model for predicting human drug metabolism.

Protective roles of cytoplasmic p21Cip1 /Waf1 in senolysis and ferroptosis of lung cancer cells.

OBJECTIVE: Therapy-induced senescent cancer cells increase the expression of the cyclin-dependent kinase inhibitors p16Ink4a and p21Cip1/Waf1 . Given that p21 regulates not only the cell cycle but also cell death, we investigated the roles of p21 in cell death using a p16-negative A549 human lung adenocarcinoma cell line. METHODS: Senescence was induced by doxorubicin (DXR) or pemetrexed (PEM). The protein expression of p21 was examined by immunoblot. Cell death, reactive oxygen species (ROS) and lipid peroxidation were determined by flow cytometry. ABT-263 and ABT-737 were used as senolytic drugs. In vivo growth of A549 cells with different levels of p21 and their sensitivity to PEM were examined in xenograft models. RESULTS: DXR-induced senescent A549 cells increased the expression of cytoplasmic p21, and the sensitivity to ABT-263 was augmented in p21-knockout A549 (A549-KOp21) cells. A similar senolytic effect was observed when PEM was combined with ABT-737. PEM alone induced a higher level of non-apoptotic cell death, ferroptosis, in A549-KOp21 cells than in A549 cells. Although there was no difference in the level of lipid peroxidation, ROS levels were higher in PEM-treated A549-KOp21 cells than in PEM-treated A549 cells. A loss of p21 increased the sensitivity of A549 cells to PEM both in vitro and in vivo. A clinical database analysis showed that CDKN1Ahigh lung adenocarcinoma patients had a poorer prognosis compared to CDKN1Alow patients. CONCLUSION: Cytoplasmic p21, which was increased in therapy-induced senescent lung cancer cells, plays protective roles in senolysis and ferroptosis.

Hydroxychloroquine Promotes Bcl-xL Inhibition-induced Apoptosis in BxPC-3 Human Pancreatic Cancer Cells.

BACKGROUND/AIM: Anti-apoptotic proteins, including Bcl-2 and Bcl-xL, hinder cancer treatment, and several drugs targeting these molecules have been developed. One is ABT-263 (navitoclax), which targets Bcl-2, Bcl-xL, and Bcl-w. On the other hand, hydroxychloroquine (HCQ) has been used as a drug for malaria infection and autoimmune disease. HCQ can exert a similar effect as chloroquine with fewer adverse events. In addition, HCQ exerts antitumor activity. In the present study, the effects of HCQ on ABT-263-induced antitumor activities were examined using three human pancreatic cancer cell lines (PANC-1, MiaPaCa-2, and BxPC-3). MATERIALS AND METHODS: In vitro effects of HCQ and ABT-263 were examined by cell viability, colony-forming assays, and flow cytometry. Protein expression was determined by immunoblotting. In vivo effects of HCQ and ABT-263 were examined by a xenograft mice model. RESULTS: Combined treatment with HCQ and ABT-263 synergistically decreased the viability of only BxPC-3 cells. This synergistic effect was not observed when HCQ was combined with ABT-199, an inhibitor specific to Bcl-2. The combination of HCQ and ABT-263 induced caspase-dependent apoptosis. Protein expression of Bcl-xL was more highly expressed in BxPC-3 cells than in the other two cell lines, and the combination of HCQ with a Bcl-xL inhibitor or siRNA-mediated knockdown of Bcl-xL induced apoptosis in BxPC-3 cells. Combination therapy with HCQ and ABT 737, an ABT-263 analogue, suppressed the in vivo growth of BxPC-3 with transient body-weight loss. CONCLUSION: HCQ effectively promotes Bcl-xL inhibition-induced apoptosis in BxPC-3 human pancreatic cancer cells.

A transchromosomic rat model with human chromosome 21 shows robust Down syndrome features.

Progress in earlier detection and clinical management has increased life expectancy and quality of life in people with Down syndrome (DS). However, no drug has been approved to help individuals with DS live independently and fully. Although rat models could support more robust physiological, behavioral, and toxicology analysis than mouse models during preclinical validation, no DS rat model is available as a result of technical challenges. We developed a transchromosomic rat model of DS, TcHSA21rat, which contains a freely segregating, EGFP-inserted, human chromosome 21 (HSA21) with >93% of its protein-coding genes. RNA-seq of neonatal forebrains demonstrates that TcHSA21rat expresses HSA21 genes and has an imbalance in global gene expression. Using EGFP as a marker for trisomic cells, flow cytometry analyses of peripheral blood cells from 361 adult TcHSA21rat animals show that 81% of animals retain HSA21 in >80% of cells, the criterion for a "Down syndrome karyotype" in people. TcHSA21rat exhibits learning and memory deficits and shows increased anxiety and hyperactivity. TcHSA21rat recapitulates well-characterized DS brain morphology, including smaller brain volume and reduced cerebellar size. In addition, the rat model shows reduced cerebellar foliation, which is not observed in DS mouse models. Moreover, TcHSA21rat exhibits anomalies in craniofacial morphology, heart development, husbandry, and stature. TcHSA21rat is a robust DS animal model that can facilitate DS basic research and provide a unique tool for preclinical validation to accelerate DS drug development.

T-cell responses and combined immunotherapy against human carbonic anhydrase 9-expressing mouse renal cell carcinoma.

Renal cell carcinoma (RCC) is known to respond to immune checkpoint blockade (ICB) therapy, whereas there has been limited analysis of T-cell responses to RCC. In this study, we utilized human carbonic anhydrase 9 (hCA9) as a model neoantigen of mouse RENCA RCC. hCA9-expressing RENCA RCC (RENCA/hCA9) cells were rejected in young mice but grew in aged mice. CD8+ T cells were the primary effector cells involved in rejection in young mice, whereas CD4+ T cells participated at the early stage. Screening of a panel of hCA9-derived peptides revealed that mouse CD8+ T cells responded to hCA9288-296 peptide. Mouse CD4+ T cells responded to lysates of RENCA/hCA9, but not RENCA cells, and showed reactivity to hCA9 276-290, which shares three amino acids with hCA9 288-296 peptide. Immunohistochemistry analysis revealed that few T cells infiltrated RENCA/hCA9 tissues in aged mice. ICB therapy of anti-PD-1/anti-CTLA-4 antibodies promoted T-cell infiltration into tumor tissues, whereas no definite antitumor effect was observed. However, additional combination with cyclophosphamide or axitinib, a vascular endothelial growth factor receptor inhibitor, induced complete regression in half of the RENCA/hCA9-bearing aged mice with increased expression of PD-L1 in tumor tissues. These results indicate that hCA9 can be a useful model neoantigen to investigate antitumor T-cell responses in mice with RCC, and that RENCA/hCA9 in aged mice can serve as a non-inflamed 'cold' tumor model facilitating the development of effective combined immunotherapies for RCC.

An improved TK-NOG mouse as a novel platform for humanized liver that overcomes limitations in both male and female animals.

We developed a novel immunodeficient NOG mouse expressing HSVtk mutant clone 30 cDNA under the control of mouse transthyretin gene enhancer/promoter (NOG-TKm30) to acquire fertility in males and high inducibility of liver injury in females. Maximum human albumin levels (approx. 15 mg/mL plasma) in both male and female NOG-TKm30 mice engrafted with human hepatocytes (humanized liver mice) were observed 8-12 weeks after transplantation. Immunohistochemical analyses revealed abundant expression of major human cytochrome P450 (CYP) enzymes (CYP1A2, CYP2C9, CYP2D6, CYP2E1, and CYP3A4) in reconstituted liver with original zonal distribution. In vivo drug-drug interactions were observed in humanized liver mice as decreased area under the curve of midazolam (CYP3A4/5 substrate) and omeprazole (CYP3A4/5 and CYP2C19 substrate) after oral administration of rifampicin. Furthermore, we developed a pregnant model for evaluating prenatal exposure to drugs. The detection of thalidomide metabolites in the fetuses of pregnant humanized liver mice indicates that the novel TK model can be used for developmental toxicity studies requiring the assessment of human drug metabolism. These results suggest that the limitations of traditional TK-NOG mice can be addressed using NOG-TKm30 mice, which constitute a novel platform for humanized liver for both in vivo and in vitro studies.

Protective role of cytoplasmic p21Cip1/Waf1 in apoptosis of CDK4/6 inhibitor-induced senescence in breast cancer cells.

Inhibition of CDK4/6 slows the cell cycle and induces senescence in breast cancer cells. However, senescent cancer cells promote invasion and metastasis. Several drugs reportedly target senescent cells, including ABT-263 (navitoclax). We examined the effects of the CDK4/6 inhibitor abemaciclib and ABT-263 on two human breast cancer cell lines. The abemaciclib and ABT-263 combination additively decreased the viability of MDA-MB-231 cells, but not MCF-7 cells. Also, the combination therapy-induced caspase-dependent apoptosis in MDA-MB-231 cells. Combination therapy with abemaciclib and ABT-737, an ABT-263 analog, significantly suppressed the in vivo growth of MDA-MB-231 with transient body-weight loss. Given that p16Ink4a and p21Cip1/Waf1 are key factors in senescence and that both cell lines were negative for p16, the role of p21 in apoptosis of treated breast cancer cells was investigated. Although abemaciclib increased the cytoplasmic p21 level in both cell lines as a hallmark of senescence, the abemaciclib and ABT-263 combination decreased it only in MDA-MB-231 cells. This decrease of p21 expression was relieved by caspase inhibition, and p21 was colocalized with caspase-3 in the cytoplasm of MDA-MB-231 cells. Alternatively, small interfering RNA-mediated knockdown of p21 rendered caspase-3-negative MCF-7 cells susceptible to abemaciclib and ABT-263, as well as TNF-related apoptosis-inducing ligand. Furthermore, a clinical database analysis showed that p21high breast cancer patients had a poorer prognosis compared to p21low patients. These results suggest that cytoplasmic p21 plays a protective role in apoptosis of CDK4/6 inhibitor-induced senescent breast cancer cells.

Immunogenic chemotherapy in two mouse colon cancer models.

Aside from the induction of cell death, some anticancer chemotherapeutic drugs can modulate antitumor immune responses. In this study, we examined the anticancer effects of 5-fluorouracil (5-FU) and oxaliplatin (L-OHP), which are standard chemotherapeutic drugs for colon cancer, combined with cyclophosphamide (CP) in two mouse colon cancer models (CT26 and MC38 colon adenocarcinoma models). In the CT26 model, two injections of 5-FU/L-OHP and CP significantly suppressed the growth of subcutaneously established CT26 tumors compared with either 5-FU/L-OHP or CP, without a significant loss of body weight. The anticancer effect was weakened in nude mice. Cured mice acquired protective immunity against CT26, and CT26-specific cytotoxic T cells (CTLs) were induced from their spleen cells. Analysis of tumor-infiltrating immune cells revealed that 5-FU/L-OHP treatment with or without CP increased the proportion of CD8+ T cells at tumor sites. The 5-FU/L-OHP treatment decreased the proportion of granulocytic myeloid-derived suppressor cells (MDSCs) and increased monocytic MDSCs in tumor sites, whereas the addition of CP treatment reversed these changes. In the MC38 model, although significant anticancer effects of the triple combination therapy were seen, additional treatment with anti-PD-1 antibody increased the number of cured mice. These mice exhibited protective immunity against MC38, and MC38-specific CTLs were generated from their spleen cells. Together, these results indicate that the antitumor effects of the combination of 5-FU/L-OHP and CP mainly depend on host T cells; moreover, the therapeutic efficacy can be effectively boosted by immune checkpoint blockade.

Local injection of CCL19-expressing mesenchymal stem cells augments the therapeutic efficacy of anti-PD-L1 antibody by promoting infiltration of immune cells.

BACKGROUND: Mesenchymal stem/stromal cells (MSC) accumulate and reside in tumor sites. METHODS: Taking advantage of this feature in anticancer therapy, immortalized murine MSC (iMSC) were genetically altered to produce chemokine (C-C motif) ligand 19 (iMSC/CCL19), which attracts dendritic cells (DC) and T lymphocytes. Thereafter, iMSC/CCL19 were examined for their therapeutic efficacy using a syngeneic CT26 colon carcinoma cell line. RESULTS: Co-injection of iMSC/CCL19 into mice significantly suppressed the in vivo growth of CT26 cells compared with that of CCL19-expressing immortalized fibroblasts (iFib/CCL19). This anticancer effect was not observed when injected in CT26-bearing nude mice. Co-injected iMSC/CCL19 survived longer than iFib/CCL19 in the tumor sites. In a therapeutic model, local injection of iMSC/CCL19 suppressed the tumor growth, and increased IFN (interferon)-γ+ CD8+ T cells and CCR7+ DC infiltration in tumor site was observed when treated with iMSC/CCL19, but not with iMSC. This antitumor effect was completely negated by depletion of CD4+ cells and partially negated by depletion of CD8+ cells. Furthermore, the antitumor effects induced by local injection of iMSC/CCL19 were augmented by additional therapy with anti-programmed death (PD)-ligand 1 (PD-L1) antibody, but not with anti-PD-1 antibody. This combination therapy cured most of the tumors in CT26-bearing mice. CONCLUSION: These results suggest that local therapy with iMSC/CCL19 can suppress tumor growth via effective recruitment of CCR7+ DC into tumor sites and increase IFN-γ+ CD8+ T cells, and that combination with anti-PD-L1 antibody therapy can be a powerful anticancer therapy.

Supplementation of l-arginine boosts the therapeutic efficacy of anticancer chemoimmunotherapy.

Myeloid-derived suppressor cells (MDSCs) play a crucial role in immunosuppression in tumor-bearing hosts. MDSCs express arginase-I and indoleamine 2,3-dioxygenase; they suppress T-cell function by reducing the levels of l-arginine and l-tryptophan, respectively. We examined the anticancer effects of supplementation of these amino acids in CT26 colon carcinoma-bearing mice. Oral supplementation of l-arginine or l-tryptophan (30 mg/mouse) did not affect tumor growth, whereas oral supplementation of d-arginine was lethal. Supplementation of l-arginine showed a tendency to augment the efficacy of cyclophosphamide (CP). CP reduced the proportions of granulocytic MDSCs and increased the proportions of monocytic MDSCs in the spleen and tumor tissues of CT26-bearing mice. l-Arginine supplementation alone did not affect the MDSC subsets. CP treatment tended to reduce the plasma levels of l-arginine in CT26-bearing mice and significantly increased the number of tumor-infiltrating CD8+ T cells. In addition, l-arginine supplementation significantly increased the proportions of tumor peptide-specific CD8+ T cells in draining lymph nodes. Importantly, additional supplementation of l-arginine significantly increased the number of cured mice that were treated with CP and anti-PD-1 antibody. Totally, l-arginine supplementation shows promise for boosting the therapeutic efficacy of chemoimmunotherapy.

Pemetrexed sensitizes human lung cancer cells to cytotoxic immune cells.

Pemetrexed (PEM) is a useful drug that can be combined with immune checkpoint blockade therapy for treatment of patients with advanced non-small-cell lung cancer (NSCLC). However, its effects on anti-cancer immunity, especially the sensitivity of NSCLC cells to cytotoxic immune cells, have not been fully investigated. In this study, we examined the effects of PEM on the sensitivity of human NSCLC cells to two different types of cytotoxic immune cells. Pre-treatment with PEM increased the sensitivity of two NSCLC cell lines, PC9 and A549, to activated T cells and natural killer (NK) cells, and decreased the expression of anti-apoptotic proteins, including XIAP and Mcl-1. In addition, PEM treatment increased the cell surface expression of programmed death-ligand 1 (PD-L1) on PC9 cells. PEM-induced upregulation of PD-L1 on PC9 cells was at least partially ascribed to activation of ERK and the NFκB pathway. In contrast, PEM treatment increased the expression of UL16-binding proteins (ULBP), ligands for the NKG2D NK receptor, on PC9 and A549 cells, as well as the induction of senescence. Although the addition of anti-programmed cell death 1 antibody showed no effect on the sensitivity of PEM-treated PC9 and A549 cells to activated T cells, that of anti-NKG2D antibody decreased the enhanced sensitivity of PEM-treated A549 cells to NK cells. These results indicate that PEM can effectively sensitize human NSCLC cells to cytotoxic immune cells while modulating the expression of immune-regulatory molecules.

Different sensitivities of senescent breast cancer cells to immune cell-mediated cytotoxicity.

Senescence is a state of growth arrest induced not only in normal cells but also in cancer cells by aging or stress, which triggers DNA damage. Despite growth suppression, senescent cancer cells promote tumor formation and recurrence by producing cytokines and growth factors; this state is designated as the senescence-associated secretory phenotype. In this study, we examined the susceptibility of senescent human breast cancer cells to immune cell-mediated cytotoxicity. Doxorubicin (DXR) treatment induced senescence in 2 human breast cancer cell lines, MDA-MB-231 and BT-549, with the induction of γH2AX expression and increased expression of p21 or p16. Treatment with DXR also induced the expression of senescence-associated ß-galactosidase and promoted the production of pro-inflammatory cytokines. Importantly, DXR-treated senescent MDA-MB-231 cells showed increased sensitivity to 2 types of immune cell-mediated cytotoxicity: cytotoxicity of activated CD4+ T cells and Ab-dependent cellular cytotoxicity by natural killer cells. This increased sensitivity to cytotoxicity was partially dependent on tumor necrosis factor-related apoptosis-inducing ligand and perforin, respectively. This increased sensitivity was not observed following treatment with the senescence-inducing cyclin-dependent kinase-4/6 inhibitor, abemaciclib. In addition, treatment with DXR, but not abemaciclib, decreased the expression of antiapoptotic proteins in cancer cells. These results indicated that DXR and abemaciclib induced senescence in breast cancer cells, but that they differed in their sensitivity to immune cell-mediated cytotoxicity. These findings could provide an indication for combining anticancer immunotherapy with chemotherapeutic drugs or molecular targeting drugs.

Bcl-2 inhibition sensitizes triple-negative human breast cancer cells to doxorubicin.

Breast cancers can be divided into several types. Because triple-negative breast cancer (TNBC) is the most refractory to current anti-cancer therapies, efficient treatment has been urgently required. Members of the Bcl-2 family play pro- and anti-apoptotic roles in mitochondria-mediated apoptosis. Some Bcl-2 family members are expressed in breast cancer and influence the response to anti-cancer therapies. In this study, we investigated whether Bcl-2 inhibition could sensitize TNBC cells to the genotoxic drug doxorubicin (DR). Treatment with a combination of the Bcl-2 inhibitor ABT-199 and DR synergistically decreased the viability of the TNBC cell lines MDA-MB-231 and BT-549. In an apoptosis assay, the combination treatment resulted in only a marginal effect in BT-549 cells, whereas drastic apoptosis was induced in MDA-MB-231 cells treated with both ABT-199 and DR. Both caspase-8 and -9 were involved in the combination treatment-induced apoptosis. Short interfering RNA-mediated knockdown of Bcl-2 increased the sensitivity of both cell lines to DR. The combination treatment also significantly decreased the colony-forming ability of the TNBC cell lines. In a xenograft mouse model, oral administration of ABT-199 augmented the DR-induced antitumor effect on subcutaneously established MDA-MB-231 cells. These results indicate that the combination of DR with Bcl-2 inhibitors, including ABT-199, may be a promising treatment modality for TNBC patients.

Chloroquine augments TRAIL-induced apoptosis and induces G2/M phase arrest in human pancreatic cancer cells.

Autophagy contributes to the treatment-resistance of many types of cancers, and chloroquine (CQ) inhibits autophagy. The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) kills cancer cells but is minimally cytotoxic to normal cells. However, because the therapeutic efficacy of TRAIL is limited, it is necessary to augment TRAIL-induced anti-tumor effects. In this study, we explored the anti-tumor effects of a combination of CQ and TRAIL on two human pancreatic cancer cell lines: TRAIL-sensitive MiaPaCa-2 cells and Panc-1 cells that are less sensitive to TRAIL. Although both CQ and TRAIL reduced cancer cell viability in a dose-dependent manner, the combination acted synergistically. CQ increased the expression level of type-II LC3B without decreasing the expression of p62, an autophagic substrate, thus indicating inhibition of autophagy. CQ did not increase the levels of death receptors on cancer cells but reduced the expression of anti-apoptotic proteins. A combination of CQ and TRAIL significantly increased cancer cell apoptosis. CQ induced cell-cycle arrest in the G2/M phase. Also, CQ increased the p21 level but reduced that of cyclin B1. A combination of CQ and TRAIL reduced the colony-forming abilities of cancer cells to extents greater than either material alone. In xenograft models, combination CQ and TRAIL therapy significantly suppressed the growth of subcutaneously established MiaPaCa-2 and Panc-1 cells, compared with the untreated or monotherapy groups. Together, the results indicate that CQ in combination with TRAIL may be useful to treat human pancreatic cancer.

Contrasting effects of cyclophosphamide on anti-CTL-associated protein 4 blockade therapy in two mouse tumor models.

Immune checkpoint blockade is a promising anticancer therapy, but must be used in combination with other anticancer therapies to increase its therapeutic efficacy. Cyclophosphamide (CP) is a chemotherapeutic drug that shows immune-modulating effects. In this study, we examined the effect of CP on anti-CTL-associated protein 4 (CTLA-4) blockade therapy in two mouse tumor models. Drastic tumor regression was observed in the CT26 colon carcinoma model after i.p. injection of CP (100 mg/kg) followed by anti-CTLA-4 antibody. However, administration in the reverse order increased apoptosis in tumor-specific CD8+ T cells. In the RENCA renal carcinoma model, the antitumor effect of combination therapy was marginal and the tumor-bearing state reduced body weight with an increased serum level of interleukin-6. Interestingly, although CP monotherapy increased myeloid-derived suppressor cells (MDSCs) in the spleens of both models, subsequent anti-CTLA-4 therapy increased MDSCs only in RENCA-bearing mice. Additionally, the serum levels of chemokine ligand 2 and C-X-C motif chemokine 10 were increased by the combination therapy only in RENCA-bearing mice and in vivo depletion of Gr-1+ cells augmented the antitumor effect to some degree. These results reveal a contrasting effect of CP on anti-CTLA-4 therapy between the two mouse tumor models. Cyclophosphamide augments the antitumor effect of anti-CTLA-4 therapy in CT26-bearing hosts, whereas CP after anti-CTLA-4 therapy attenuates this effect through induction of apoptosis in tumor-reactive T cells. Alternatively, CP-induced MDSCs can be increased by anti-CTLA-4 therapy only in RENCA-bearing hosts with an elevated level of interleukin-6.

Bi-HAC vector system toward gene and cell therapy.

Genetic manipulations with mammalian cells often require introduction of two or more genes that have to be in trans-configuration. However, conventional gene delivery vectors have several limitations, including a limited cloning capacity and a risk of insertional mutagenesis. In this paper, we describe a novel gene expression system that consists of two differently marked HAC vectors containing unique gene loading sites. One HAC, 21HAC, is stably propagated during cell divisions; therefore, it is suitable for complementation of a gene deficiency. The other HAC, tet-O HAC, can be eliminated, providing a unique opportunity for transient gene expression (e.g., for cell reprogramming). Efficiency and accuracy of a novel bi-HAC vector system have been evaluated after loading of two different transgenes into these HACs. Based on analysis of transgenes expression and HACs stability in the proof of principle experiments, the combination of two HAC vectors may provide a powerful tool toward gene and cell therapy.

A novel and stable mouse artificial chromosome vector.

Human chromosome fragments (hCFs) and human artificial chromosomes (HACs) can be transferred into mouse ES cells to produce trans-chromosomic (Tc) mice. Although hCFs and HACs containing large genomic DNAs can be autonomously maintained in Tc mice, their retention rate is variable in mouse ES cell lines and Tc mouse tissues, possibly because of centromere differences between the species. To improve the retention rate of artificial chromosomes in mouse cells, we constructed novel mouse artificial chromosome (MAC) vectors by truncating a natural mouse chromosome at a site adjacent to the centromeric region. We obtained cell clones containing the MAC vectors that were stably maintained in mouse ES cells and various tissues in Tc mice. The MACs possess acceptor sites into which a desired gene or genes can be inserted. Thus, Tc mice harboring the MAC vectors may be valuable tools for functional analyses of desired genes, producing humanized model mice, and synthetic biology.