Autophagy inhibition switches low-dose camptothecin-induced premature senescence to apoptosis in human colorectal cancer cells.

Recently, several studies indicated that senescent tumor cells are resistant to apoptosis in chemotherapy. They may return to cell cycle, thus act as stumbling blocks in anticancer treatments. In the present study, we found that, in human colorectal cancer cells, low-dose camptothecin (CPT) simultaneously induced autophagy and premature senescence through AMPK-TSC2-mTOR pathway and ATM-Chk2-p53-p21 pathway respectively. What's important is the suppression of autophagy substantially increased apoptosis and greatly attenuated senescence possibly by blocking p53/p21 pathway, which suggests that autophagy plays an indispensable role in sustaining cell senescence caused by low-dose CPT. The combination of low-dose CPT and autophagy inhibitor, a way to lead senescent cells to die, would be potentially valuable in cancer therapy.

Bortezomib induces protective autophagy through AMP-activated protein kinase activation in cultured pancreatic and colorectal cancer cells.

BACKGROUND: Bortezomib, a selective and potent inhibitor of the proteasome, has demonstrated broad anti-tumor activities in many malignancies. In the current study, we aimed to understand the potential resistance factor of bortezomib in cultured pancreatic and colorectal cancer cells. RESULTS: We observed that bortezomib-induced protective autophagy in cultured PANC-1 pancreatic cancer cells and HT-29 colorectal cancer cells. Inhibition of autophagy by 3-methyladenine (3-MA) and chloroquine enhanced bortezomib-induced apoptosis and cytotoxicity in both PANC-1 and HT-29 cells. Activation of AMP-activated protein kinase (AMPK) was required for bortezomib-induced autophagy induction in PANC-1 and HT-29 cells, and AMPK inhibition by its inhibitor compound C (CC) or RNAi-depletion suppressed bortezomib-induced autophagy, while dramatically enhancing cancer cell apoptosis/cytotoxicity. Meanwhile, significant AMPK activation and autophagy induction were observed after bortezomib stimulation in primary cultured pancreatic cancer cells derived from a patient's tumor tissue. Both CC and 3-MA facilitated bortezomib-induced cytotoxicity in primary cultured pancreatic cancer cells. CONCLUSIONS: In conclusion, our data here suggest that bortezomib induces protective autophagy in pancreatic and colorectal cancer cells through activating AMPK-Ulk1 signalings. AMPK or autophagy inhibitors could be developed as an adjunct or chemo-sensitizer for bortezomib.

Regulation of death induction and chemosensitizing action of 3-bromopyruvate in myeloid leukemia cells: energy depletion, oxidative stress, and protein kinase activity modulation.

3-Bromopyruvate (3-BrP) is an alkylating, energy-depleting drug that is of interest in antitumor therapies, although the mechanisms underlying its cytotoxicity are ill-defined. We show here that 3-BrP causes concentration-dependent cell death of HL60 and other human myeloid leukemia cells, inducing both apoptosis and necrosis at 20-30 µM and a pure necrotic response at 60 µM. Low concentrations of 3-BrP (10-20 µM) brought about a rapid inhibition of glycolysis, which at higher concentrations was followed by the inhibition of mitochondrial respiration. The combination of these effects causes concentration-dependent ATP depletion, although this cannot explain the lethality at intermediate 3-BrP concentrations (20-30 µM). The oxidative stress caused by exposure to 3-BrP was evident as a moderate overproduction of reactive oxygen species and a concentration-dependent depletion of glutathione, which was an important determinant of 3-BrP toxicity. In addition, 3-BrP caused glutathione-dependent stimulation of p38 mitogen-activated protein kinase (MAPK), mitogen-induced extracellular kinase (MEK)/extracellular signal-regulated kinase (ERK), and protein kinase B (Akt)/mammalian target of rapamycin/p70S6K phosphorylation or activation, as well as rapid LKB-1/AMP kinase (AMPK) activation, which was later followed by Akt-mediated inactivation. Experiments with pharmacological inhibitors revealed that p38 MAPK activation enhances 3-BrP toxicity, which is conversely restrained by ERK and Akt activity. Finally, 3-BrP was seen to cooperate with antitumor agents like arsenic trioxide and curcumin in causing cell death, a response apparently mediated by both the generation of oxidative stress induced by 3-BrP and the attenuation of Akt and ERK activation by curcumin. In summary, 3-BrP cytotoxicity is the result of several combined regulatory mechanisms that might represent important targets to improve therapeutic efficacy.

Arsenic trioxide co-exposure potentiates benzo(a)pyrene genotoxicity by enhancing the oxidative stress in human lung adenocarcinoma cell.

Although both arsenic trioxide (As2O3) and benzo(a)pyrene (BaP) are well-established human carcinogens, the interaction between As2O3 and BaP is synergistic or antagonistic remains controversial in terms of the existing studies. In addition, the mechanisms responsible for the combined effects are still unclear. In this study, we examined the potential interactive effects between As2O3 (1, 5, and 10 µM) and BaP (5, 10, and 20 µM) in cultured A549 cells by treating with BaP and As2O3 alone or in combination at various concentrations for 24 h. The single and combined effects of As2O3 and BaP on the cytotoxicity, DNA/chromosomal damage, and oxidative stress were examined by using tetrazolium (3-(4,5-dimethyithiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) dye colorimetric assay, colony formation assay, fluorescence probe, chemical colorimetry, comet assay as well as micronucleus test. Our results showed that As2O3 synergistically enhanced the cytotoxicity, genotoxicity, and level of oxidative stress induced by BaP at various tested concentrations. Also, our experimental results showed that intracellular glutathione (GSH) contents were increased by various doses of BaP, but single or cotreatment with As2O3 significantly decreased the GSH level in the cells at all tested concentrations. Taken together, our results suggest that As2O3 may exert its synergistic cyto- and genotoxic effects with BaP mainly via elevated intracellular reactive oxygen species and reduced GSH contents and superoxide dismutase activities, thus promoting high level of oxidative stress, which may be a pivotal mechanism underlying As2O3 cocarcinogenic action.

PD173074, a selective FGFR inhibitor, reverses ABCB1-mediated drug resistance in cancer cells.

PURPOSE: Specific tyrosine kinase inhibitors were recently reported to modulate the activity of ABC transporters, leading to an increase in the intracellular concentration of their substrate drugs. In this study, we determine whether PD173074, a specific fibroblast growth factor receptor (FGFR) inhibitor, could reverse ABC transporter-mediated multidrug resistance. METHODS: 3-(4,5-Dimethylthiazol-yl)-2,5-diphenyllapatinibrazolium bromide assay was used to determine the effect of PD173074 on reversal of ABC transporter-mediated multidrug resistance (MDR). In addition, [³H]-paclitaxel accumulation/efflux assay, western blotting analysis, ATPase, and photoaffinity labeling assays were done to study the interaction of PD173074 on ABC transporters. RESULTS: PD173074 significantly sensitized both ABCB1-transfected and drug-selected cell lines overexpressing this transporter to substrate anticancer drugs colchicine, paclitaxel, and vincristine. This effect of PD173074 is specific to ABCB1, as no significant interaction was detected with other ABC transporters such as ABCC1 and ABCG2. The observed reversal effect seems to be primarily due to the decreased active efflux of [³H]-paclitaxel in ABCB1 overexpressing cells observed in efflux assay. In addition, no significant change in the ABCB1 expression was observed when ABCB1 overexpressing cells were exposed to 5 µM PD173074 for up to 3 days, thereby further suggesting its role in modulating the function of the transporter. In addition, PD173074 stimulated the ATPase activity of ABCB1 in a concentration-dependent manner, indicating a direct interaction with the transporter. Interestingly, PD173074 did not inhibit photolabeling of ABCB1 with [¹²5I]-iodoarylazidoprazosin (IAAP), showing that it binds at a site different from that of IAAP in the drug-binding pocket. CONCLUSIONS: Here, we report for the first time, PD173074, an inhibitor of the FGFR, to selectively reverse ABCB1 transporter-mediated MDR by directly blocking the efflux function of the transporter.

Metformin enhances cisplatin cytotoxicity by suppressing signal transducer and activator of transcription-3 activity independently of the liver kinase B1-AMP-activated protein kinase pathway.

Metformin has been used as first-line treatment in patients with type 2 diabetes, and is reported to reduce cancer risk and progression by activating the liver kinase B1 (LKB1)-AMP-activated protein kinase (AMPK) pathway. Cisplatin remains the main drug for treating advanced non-small-cell lung cancer. However, drug resistance often develops through several mechanisms during the treatment course, including one mechanism mediated by the activation of the IL-6/signal transducer and activator of transcription (STAT)-3 pathway, related to the generation of reactive oxygen species (ROS). This study demonstrated a correlation between STAT3 phosphorylation and cisplatin cytotoxicity, using AS2 (PC14PE6/AS2)-derived cell lines (AS2/S3C) that contained constitutively active STAT3 plasmids as a model. A STAT3 inhibitor (JSI-124) enhanced the cisplatin sensitivity in AS2 cells, whereas metformin inhibited STAT3 phosphorylation and enhanced cisplatin cytotoxicity. By contrast, another AMPK activator (5-aminoimidazole-4-carboxamide-riboside) failed to produce these effects. LKB1-AMPK silencing by small, interfering RNA or mammalian target of rapamycin (mTOR) inhibition by rapamycin or pp242 did not alter the effect of metformin on STAT3 activity suppression, suggesting that metformin can modulate the STAT3 pathway through an LKB1-AMPK-independent and probably mTOR-independent mechanism. Metformin also inhibited cisplatin-induced ROS production and autocrine IL-6 secretion in AS2 cells. Both mechanisms contributed to the ability of metformin to suppress STAT3 activation in cancer cells, which resulted in the decreased secretion of vascular endothelial growth factor by cancer cells. The growth of subcutaneous tumor xenografts was significantly delayed by a combination of cisplatin and metformin. This is the first study to demonstrate that metformin suppresses STAT3 activation via LKB1-AMPK-mTOR-independent but ROS-related and autocrine IL-6 production-related pathways. Thus, metformin helps to overcome tumor drug resistance by targeting STAT3.

Monoclonal antibodies: potential new therapeutic treatment against multiple myeloma.

Despite recent treatments, such as bortezomib, thalidomide, and lenalidomide, therapy of multiple myeloma (MM) is limited, and MM remains an incurable disease associated with high mortality. The outcome of patients treated with cytotoxic therapy has not been satisfactory. Therefore, new therapies are needed for relapsed MM. A new anticancer strategy is the use of monoclonal antibodies (MoAbs) that represent the best available combination of tumor cytotoxicity, environmental signal privation, and immune system redirection. Clinical results in patients with relapsed/refractory MM suggest that MoAbs are likely to operate synergistically with traditional therapies (dexamethasone), immune modulators (thalidomide, lenalidomide), and other novel therapies (bortezomib); in addition, MoAbs have shown the ability to overcome resistance to these therapies. It remains to be defined how MoAb therapy can most fruitfully be incorporated into the current therapeutic paradigms that have achieved significant survival earnings in patients with MM. This will require careful consideration of the optimal sequence of treatments and their clinical position as either short-term induction therapy, frontline therapy in patients ineligible for ASCT, or long-term maintenance treatment.

The sorafenib plus nutlin-3 combination promotes synergistic cytotoxicity in acute myeloid leukemic cells irrespectively of FLT3 and p53 status.

BACKGROUND: Both the multi-kinase inhibitor sorafenib and the small molecule inhibitor of the MDM2/p53 interaction, nutlin-3, used alone, have shown promising anti-leukemic activity in acute myeloid leukemia cells. Thus, in this study we investigated the effect of the combination of sorafenib plus nutlin-3 in acute myeloid leukemia. DESIGN AND METHODS: Primary acute myeloid leukemia blasts (n=13) and FLT3(wild-type)/p53(wild-type) (OCI-AML3), FLT3(mutated)/p53(wild-type) (MOLM), FLT3(mutated)/p53(mutated) (MV4-11), FLT3(wild-type)/p53(deleted) (HL60) or FLT3(wild-type)/p53(mutated) (NB4) acute myeloid cell lines were exposed to sorafenib, used alone or in association with nutlin-3 at a 1:1 ratio, in a range of clinically achievable concentrations (1-10 µM). Induction of apoptosis and autophagy was evaluated by transmission electron microscopy and by specific flow cytometry analyses. The levels of Mcl-1, p53 and Bak proteins were analyzed by western blotting. Knock-down of Bax and Bak gene expression was performed in transfection experiments with specific short interfering RNA. RESULTS: The sorafenib+nutlin-3 drug combination exhibits synergistic cytotoxicity in primary acute myeloid leukemia blasts and in acute myeloid leukemia cell lines with maximal cytotoxicity in FLT3(mutated) MV4-11 and MOLM, followed by the FLT3(wild-type) OCI-AML3, HL60 and NB4 cell lines. The cytotoxic activity of sorafenib+nutlin-3 was characterized by an increase of both apoptosis and autophagy. Moreover, Bax and Bak showed prominent roles in mediating the decrease of cell viability in response to the drug combination in p53(wild-type) OCI-AML3 and p53(deleted) HL-60 cells, respectively, as demonstrated in transfection experiments performed with specific short interfering RNA. CONCLUSIONS: Our data demonstrate that acute myeloid leukemia cells show a variable but overall good susceptibility to the innovative therapeutic combination of sorafenib+nutlin-3, which differentially involves the pro-apoptotic Bcl-2 family members Bax and Bak in p53(wild-type) and p53(deleted) cells.

Distinct roles of different forms of vitamin E in DHA-induced apoptosis in triple-negative breast cancer cells.

SCOPE: Docosahexaenoic acid (DHA) has been shown to exhibit anticancer actions in vitro and in vivo in a variety of cancers. Here, we investigated the role for DHA in inducing apoptosis in triple-negative breast cancer (TNBC) and studied the mechanisms of action. METHODS AND RESULTS: DHA induces apoptosis as detected by Annexin V-FITC/PI assay as well as induces cleavage of caspase-8 and -9, endoplasmic reticulum stress (ERS), and elevated levels of death receptor-5 (DR5) protein expression as detected by western blot assays. Chemical inhibitors of caspase-8 and -9 and small interfering RNAs (siRNAs) show DHA to induce ERS/CHOP/DR5-mediated caspase-8 and -9 dependent apoptosis. Furthermore, DHA induces elevated cellular levels of reactive oxygen species (ROS) and antioxidant; RRR-α-tocopherol (αT) blocked DHA-induced apoptotic events. In contrast to the antagonistic impact of αT, gamma-tocotrienol (γT3) was demonstrated to cooperate with DHA in inducing apoptotic events in TNBC cells. CONCLUSION: Data, for the first time, demonstrate that DHA induces apoptosis in TNBC cells via activation of ERS/CHOP/DR5-mediated caspase-8 and -9 dependent pro-apoptotic events, and that different forms of vitamin E exhibit distinct effects on DHA-induced apoptosis; namely, inhibition by αT and enhancement by γT3.

Halofuginone inhibits multiple myeloma growth in vitro and in vivo and enhances cytotoxicity of conventional and novel agents.

Multiple Myeloma (MM), a malignancy of plasma cells, remains incurable despite the use of conventional and novel therapies. Halofuginone (HF), a synthetic derivative of quinazolinone alkaloid, has recently been shown to have anti-cancer activity in various preclinical settings. This study demonstrated the anti-tumour activity of HF against a panel of human MM cell lines and primary patient-derived MM cells, regardless of their sensitivity to conventional therapy or novel agents. HF showed anti-MM activity in vivo using a myeloma xenograft mouse model. HF suppressed proliferation of myeloma cells alone and when co-cultured with bone marrow stromal cells. Similarly, HF induced apoptosis in MM cells even in the presence of insulin-like growth factor 1 or interleukin 6. Importantly, HF, even at high doses, did not induce cytotoxicity against CD40 activated peripheral blood mononuclear cells from normal donors. HF treatment induced accumulation of cells in the G(0) /G(1) cell cycle and induction of apoptotic cell death associated with depletion of mitochondrial membrane potential; cleavage of poly (ADP-ribose) polymerase and caspases-3, 8 and 9 as well as down-regulation of anti-apoptotic proteins including Mcl-1 and X-IAP. Multiplex analysis of phosphorylation of diverse components of signalling cascades revealed that HF induced changes in P38MAPK activation; increased phosphorylation of c-jun, c-jun NH(2)-terminal kinase (JNK), p53 and Hsp-27. Importantly, HF triggered synergistic cytotoxicity in combination with lenalidomide, melphalan, dexamethasone, and doxorubicin. Taken together, these preclinical studies provide the preclinical framework for future clinical studies of HF in MM.

Lycopene and the LXRα agonist T0901317 synergistically inhibit the proliferation of androgen-independent prostate cancer cells via the PPARγ-LXRα-ABCA1 pathway.

In our previous study, we demonstrated that lycopene can inhibit the proliferation of androgen-dependent prostate LNCaP cancer cells through the activation of the peroxisome proliferator-activated receptor gamma (PPARγ)-liver X receptor alpha (LXRα)-ATP-binding cassette transporter 1 (ABCA1) pathway. However, it is still unclear whether lycopene possesses similar effects in androgen-independent prostate cancer cells DU145 and PC-3. As lycopene inhibited the proliferation of both cell types to a similar extent, we chose DU145 cells for most of the subsequent studies. We show that lycopene significantly increased protein and mRNA expression of PPARγ, LXRα and ABCA1 and cholesterol efflux (i.e., decreased cellular cholesterol and increased cholesterol in culture medium). Lycopene (10 µM) in the presence of a specific antagonist of PPARγ (GW9662) or of LXRα (GGPP) restored the proliferation of DU145 cells and significantly suppressed lycopene-induced protein and mRNA expression of PPARγ and LXRα and cholesterol efflux. Liver X receptor α knockdown by siRNA against LXRα significantly promoted the proliferation of DU145 cells, whereas si-LXRα knockdown followed by incubation with lycopene (10 µM) restored the proliferation to the control level. Furthermore, lycopene in combination with the LXRα agonist T0901317 exhibited synergistic effects on cell proliferation and protein expression of PPARγ, LXRα and ABCA1. These results demonstrate that lycopene can inhibit DU145 cell proliferation via PPARγ-LXRα-ABCA1 pathway and that lycopene and T0901317 exhibit synergistic effects.

Induction of apoptosis by survivin silencing through siRNA delivery in a human breast cancer cell line.

Post-transcriptional silencing of antiapoptotic genes is a promising strategy for cancer therapy, but delivering short interfering RNA (siRNA) molecules against such targets is challenging due to inability of anionic siRNA to cross cellular membranes. Lipid substitution on small molecular weight, nontoxic polyethylenimine (PEI) has been investigated as a promising approach for effective siRNA delivery. In this study, we report on the ability of low molecular weight, lipid-substituted PEI to deliver siRNA against the antiapoptotic protein survivin. Toxicity of a library of lipid-substituted PEIs, as well as their siRNA delivery and survivin silencing efficiency, was evaluated in MDA-MB-231 human breast cancer cells. A significant increase in cellular delivery of siRNA was observed as a result of lipid substitution. Most significant downregulation of survivin was established by caprylic acid-substituted polymers, which resulted in significant levels of apoptosis induction and resultant loss of cell viability. Survivin downregulation prior to anticancer drug treatment decreased the IC(50) of several drugs by 50- to 120-fold. Our experiments indicated an effective downregulation of survivin, a cell protective protein upregulated in tumor cells, by delivering siRNA with hydrophobically modified PEI. This study introduces a promising delivery system for safe and effective siRNA delivery that will be suitable for further investigation in preclinical animal models.

Synergistic induction of apoptosis in brain cancer cells by targeted codelivery of siRNA and anticancer drugs.

Multiple dysregulated pathways in tumors necessitate targeting multiple oncogenic elements by combining orthogonal therapeutic moieties like short-interfering RNAs (siRNA) and drug molecules in order to achieve a synergistic therapeutic effect. In this manuscript, we describe the synthesis of cyclodextrin-modified dendritic polyamines (DexAMs) and their application as a multicomponent delivery vehicle for translocating siRNA and anticancer drugs. The presence of ß-cyclodextrins in our DexAMs facilitated complexation and intracellular uptake of hydrophobic anticancer drugs, suberoylanilide hydroxamic acid (SAHA) and erlotinib, whereas the cationic polyamine backbone allowed for electrostatic interaction with the negatively charged siRNA. The DexAM complexes were found to have minimal cytotoxicity over a wide range of concentrations and were found to efficiently deliver siRNA, thereby silencing the expression of targeted genes. As a proof of concept, we demonstrated that upon appropriate modification with targeting ligands, we were able to simultaneously deliver multiple payloads--siRNA against oncogenic receptor, EGFRvIII and anticancer drugs (SAHA or erlotinib)--efficiently and selectively to glioblastoma cells. Codelivery of siRNA-EGFRvIII and SAHA/erlotinib in glioblastoma cells was found to significantly inhibit cell proliferation and induce apoptosis, as compared to the individual treatments.

Cholesterol sequestration by nystatin enhances the uptake and activity of endostatin in endothelium via regulating distinct endocytic pathways.

Specific internalization of endostatin into endothelial cells has been proved to be important for its biologic functions. However, the mechanism of endostatin internalization still remains elusive. In this study, we report for the first time that both caveolae/lipid rafts and clathrin-coated pits are involved in endostatin internalization. Inhibition of either the caveolae pathway or the clathrin pathway with the use of chemical inhibitors, small interfering RNAs, or dominant-negative mutants alters endostatin internalization in vitro. Intriguingly, cholesterol sequestration by nystatin, a polyene antifungal drug, significantly enhances endostatin uptake by endothelial cells through switching endostatin internalization predominantly to the clathrin-mediated pathway. Nystatin-enhanced internalization of endostatin also increases its inhibitory effects on endothelial cell tube formation and migration. More importantly, combined treatment with nystatin and endostatin selectively enhances endostatin uptake and biodistribution in tumor blood vessels and tumor tissues but not in normal tissues of tumor-bearing mice, ultimately resulting in elevated antiangiogenic and antitumor efficacies of endostatin in vivo. Taken together, our data show a novel mechanism of endostatin internalization and support the potential application of enhancing the uptake and therapeutic efficacy of endostatin via regulating distinct endocytic pathways with cholesterol-sequestering agents.

Synergistic interactions between heregulin and peroxisome proliferator-activated receptor-gamma (PPARgamma) agonist in breast cancer cells.

Here, we demonstrate that troglitazone (Rezulin), a peroxisome proliferator-activated receptor agonist, acted in synergy with heregulin to induce massive cell death in breast cancer cells. Although the combination of heregulin and troglitazone (HRG/TGZ) induced both apoptosis and necrosis, the main mode of cell death was caspase-independent and occurred via necrosis. This combination increased generation of superoxide in mitochondria, which in turn destabilized mitochondria potential. Pretreatment with N-acetyl-l-cysteine and catalase expression ameliorated cell death induced by the combination treatment, indicating a role of oxidative stress in mediating HRG/TGZ-induced cell death. Notably, pretreatment with pyruvate significantly prevented the cell death, suggesting a potential mechanistic link between metabolic stress and HRG/TGZ-induced cell death. The activation of the HRG signaling axis has been considered as a poor prognostic factor in breast cancer and confers resistance to gefitinib (Iressa) and tamoxifen. However, our data presented here paradoxically suggest that HRG expression can actually be beneficial when it comes to treating breast cancer with peroxisome proliferator-activated receptor-γ ligands. Taken together, the combination of HRG and TGZ may provide a basis for the development of a novel strategy in the treatment of apoptosis-resistant and/or hormone-refractory breast cancer.

TLR1/TLR2 agonist induces tumor regression by reciprocal modulation of effector and regulatory T cells.

Using TLR agonists in cancer treatment can have either beneficial or detrimental effects. Therefore, it is important to determine their effect on the tumor growth and understand the underlying mechanisms in animal tumor models. In this study, we report a general immunotherapeutic activity of a synthetic bacterial lipoprotein (BLP), a TLR1/TLR2 agonist, on established lung carcinoma, leukemia, and melanoma in mice. Systemic treatment of 3LL tumor-bearing mice with BLP, but not LPS, led to a dose-dependent tumor regression and a long-lasting protective response against tumor rechallenge. The BLP-mediated tumor remission was neither mediated by a direct tumoricidal activity nor by innate immune cells, because it lacked therapeutic effect in immunodeficient SCID mice. Instead, BLP treatment reduced the suppressive function of Foxp3(+) regulatory T cells (Tregs) and enhanced the cytotoxicity of tumor-specific CTL in vitro and in vivo. Furthermore, adoptive cotransfer of BLP-pretreated but not untreated CTL and Tregs from wild-type but not from TLR2(-/-) mice was sufficient to restore antitumor immunity in SCID mice by reciprocally modulating Treg and CTL function. These results demonstrate that the TLR1/TLR2 agonist BLP may have a general tumor therapeutic property involving reciprocal downregulation of Treg and upregulation of CTL function. This property may play an important role in the development of novel antitumor strategies.

Lucanthone is a novel inhibitor of autophagy that induces cathepsin D-mediated apoptosis.

Cellular stress induced by nutrient deprivation, hypoxia, and exposure to many chemotherapeutic agents activates an evolutionarily conserved cell survival pathway termed autophagy. This pathway enables cancer cells to undergo self-digestion to generate ATP and other essential biosynthetic molecules to temporarily avoid cell death. Therefore, disruption of autophagy may sensitize cancer cells to cell death and augment chemotherapy-induced apoptosis. Chloroquine and its analog hydroxychloroquine are the only clinically relevant autophagy inhibitors. Because both of these agents induce ocular toxicity, novel inhibitors of autophagy with a better therapeutic index are needed. Here we demonstrate that the small molecule lucanthone inhibits autophagy, induces lysosomal membrane permeabilization, and possesses significantly more potent activity in breast cancer models compared with chloroquine. Exposure to lucanthone resulted in processing and recruitment of microtubule-associated protein 1 light chain 3 (LC3) to autophagosomes, but impaired autophagic degradation as revealed by transmission electron microscopy and the accumulation of p62/SQSTM1. Microarray analysis, qRT-PCR, and immunoblotting determined that lucanthone stimulated a large induction in cathepsin D, which correlated with cell death. Accordingly, knockdown of cathepsin D reduced lucanthone-mediated apoptosis. Subsequent studies using p53(+/+) and p53(-/-) HCT116 cells established that lucanthone induced cathepsin D expression and reduced cancer cell viability independently of p53 status. In addition, lucanthone enhanced the anticancer activity of the histone deacetylase inhibitor vorinostat. Collectively, our results demonstrate that lucanthone is a novel autophagic inhibitor that induces apoptosis via cathepsin D accumulation and enhances vorinostat-mediated cell death in breast cancer models.

A critical role for mast cells and mast cell-derived IL-6 in TLR2-mediated inhibition of tumor growth.

Several TLR agonists are effective in tumor immunotherapy, but their early innate mechanisms of action, particularly those of TLR2 agonists, are unclear. Mast cells are abundant surrounding solid tumors where they are often protumorigenic and enhance tumor angiogenesis. However, antitumor roles for mast cells have also been documented. The impact of mast cells may be dependent on their activation status and mediator release in different tumors. Using an orthotopic melanoma model in wild-type C57BL/6 and mast cell-deficient Kit(W-sh/W-sh) mice and a complementary Matrigel-tumor model in C57BL/6 mice, mast cells were shown to be crucial for TLR2 agonist (Pam(3)CSK(4))-induced tumor inhibition. Activation of TLR2 on mast cells reversed their well-documented protumorigenic role. Tumor growth inhibition after peritumoral administration of Pam(3)CSK(4) was restored in Kit(W-sh/W-sh) mice by local reconstitution with wild-type, but not TLR2-deficient, mast cells. Mast cells secrete multiple mediators after Pam(3)CSK(4) activation, and in vivo mast cell reconstitution studies also revealed that tumor growth inhibition required mast cell-derived IL-6, but not TNF. Mast cell-mediated anticancer properties were multifaceted. Direct antitumor effects in vitro and decreased angiogenesis and recruitment of NK and T cells in vivo were observed. TLR2-activated mast cells also inhibited the growth of lung cancer cells in vivo. Unlike other immune cells, mast cells are relatively radioresistant making them attractive candidates for combined treatment modalities. This study has important implications for the design of immunotherapeutic strategies and reveals, to our knowledge, a novel mechanism of action for TLR2 agonists in vivo.

Identification and characterization of steroidogenic factor-1 inverse agonists.

The transcription factor Steroidogenic Factor-1 (Ad4BP/SF-1; NR5A1 according to the standard nomenclature) has an essential role in adrenogonadal development. Furthermore, SF-1 is amplified and overexpressed in most cases of adrenocortical tumor occurring in children; studies performed in transgenic mice have shown that an increased SF-1 dosage triggers tumor formation in the adrenal cortex. For these reasons, drugs interfering with SF-1 action would represent a promising tool to be added to the current pharmacological protocols in the therapy of adrenocortical cancer. Here, we describe the methods how isoquinolinone compounds inhibiting the constitutive transcriptional activity of SF-1 (SF-1 inverse agonists) were identified and characterized. These compounds have the attributes to inhibit the increase in proliferation triggered by an augmented SF-1 dosage in adrenocortical tumor cells and to reduce their steroid production. This latter property may also reveal beneficial for drugs used in the therapy of adrenocortical tumors to alleviate symptoms of virilization and Cushing often associated with tumor burden.

Mullerian inhibiting substance preferentially inhibits stem/progenitors in human ovarian cancer cell lines compared with chemotherapeutics.

Cancer stem cells are proposed to be tumor-initiating cells capable of tumorigenesis, recurrence, metastasis, and drug resistance, and, like somatic stem cells, are thought to be capable of unlimited self-renewal and, when stimulated, proliferation and differentiation. Here we select cells by expression of a panel of markers to enrich for a population with stem cell-like characteristics. A panel of eight was initially selected from 95 human cell surface antigens as each was shared among human ovarian primary cancers, ovarian cancer cell lines, and normal fimbria. A total of 150 combinations of markers were reduced to a panel of three--CD44, CD24, and Epcam--which selected, in three ovarian cancer cell lines, those cells which best formed colonies. Cells expressing CD44, CD24, and Epcam exhibited stem cell characteristics of shorter tumor-free intervals in vivo after limiting dilution, and enhanced migration in invasion assays in vitro. Also, doxorubicin, cisplatin, and paclitaxel increased this enriched population which, conversely, was significantly inhibited by Müllerian inhibiting substance (MIS) or the MIS mimetic SP600125. These findings demonstrate that flow cytometry can be used to detect a population which shows differential drug sensitivity, and imply that treatment of patients can be individualized to target both stem/progenitor cell enriched and nonenriched subpopulations. The findings also suggest that this population, amenable to isolation by flow cytometry, can be used to screen for novel treatment paradigms, including biologic agents such as MIS, which will improve outcomes for patients with ovarian cancer.