Mitochondrial complex I inhibitors suppress tumor growth through concomitant acidification of the intra- and extracellular environment.

The disruption of the tumor microenvironment (TME) is a promising anti-cancer strategy, but its effective targeting for solid tumors remains unknown. Here, we investigated the anti-cancer activity of the mitochondrial complex I inhibitor intervenolin (ITV), which modulates the TME independent of energy depletion. By modulating lactate metabolism, ITV induced the concomitant acidification of the intra- and extracellular environment, which synergistically suppressed S6K1 activity in cancer cells through protein phosphatase-2A-mediated dephosphorylation via G-protein-coupled receptor(s). Other complex I inhibitors including metformin and rotenone were also found to exert the same effect through an energy depletion-independent manner as ITV. In mouse and patient-derived xenograft models, ITV was found to suppress tumor growth and its mode of action was further confirmed. The TME is usually acidic owing to glycolytic cancer cell metabolism, and this condition is more susceptible to complex I inhibitors. Thus, we have demonstrated a potential treatment strategy for solid tumors.

A novel monoclonal antibody targeting coxsackie virus and adenovirus receptor inhibits tumor growth in vivo.

To create a new anti-tumor antibody, we conducted signal sequence trap by retrovirus-meditated expression method and identified coxsackie virus and adenovirus receptor (CXADR) as an appropriate target. We developed monoclonal antibodies against human CXADR and found that one antibody (6G10A) significantly inhibited the growth of subcutaneous as well as orthotopic xenografts of human prostate cancer cells in vivo. Furthermore, 6G10A also inhibited other cancer xenografts expressing CXADR, such as pancreatic and colorectal cancer cells. Knockdown and overexpression of CXADR confirmed the dependence of its anti-tumor activity on CXADR expression. Our studies of its action demonstrated that 6G10A exerted its anti-tumor activity primarily through both antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. Moreover, 6G10A reacted with human tumor tissues, such as prostate, lung, and brain, each of which express CXADR. Although we need further evaluation of its reactivity and safety in human tissues, our results show that a novel anti-CXADR antibody may be a feasible candidate for cancer immunotherapy.

Stromal cells positively and negatively modulate the growth of cancer cells: stimulation via the PGE2-TNFα-IL-6 pathway and inhibition via secreted GAPDH-E-cadherin interaction.

Fibroblast-like stromal cells modulate cancer cells through secreted factors and adhesion, but those factors are not fully understood. Here, we have identified critical stromal factors that modulate cancer growth positively and negatively. Using a cell co-culture system, we found that gastric stromal cells secreted IL-6 as a growth and survival factor for gastric cancer cells. Moreover, gastric cancer cells secreted PGE2 and TNFα that stimulated IL-6 secretion by the stromal cells. Furthermore, we found that stromal cells secreted glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Extracellular GAPDH, or its N-terminal domain, inhibited gastric cancer cell growth, a finding confirmed in other cell systems. GAPDH bound to E-cadherin and downregulated the mTOR-p70S6 kinase pathway. These results demonstrate that stromal cells could regulate cancer cell growth through the balance of these secreted factors. We propose that negative regulation of cancer growth using GAPDH could be a new anti-cancer strategy.

New metastatic model of human small-cell lung cancer by orthotopic transplantation in mice.

Small-cell lung cancer (SCLC) is an aggressive cancer with high metastatic ability and novel strategies against the metastasis are urgently needed to improve SCLC treatment. However, the mechanism of metastasis of SCLC remains largely to be elucidated. For further studies of SCLC metastasis, we developed a new orthotopic transplantation model in mice. We established a GFP-labeled subline from the human SCLC cell line DMS273 and transplanted them orthotopically into the lung of nude mice with Matrigel. The GFP-labeled cells showed significant metastatic activity and formed metastatic foci in distant tissues such as bone, kidney, and brain, as observed in SCLC patients. From a bone metastasis focus of the mouse, we isolated another subline, termed G3H, with enhanced metastatic potential and higher hepatocyte growth factor (HGF) expression than the parental line. Further studies indicated that the HGF/MET signaling pathway was involved in in vitro motility and invasion activities of the G3H cells and treatments with MET inhibitors decreased formation of distant metastases in our orthotopic model using G3H cells. These data indicated that our model mimics the clinical aspect of SCLC such as metastatic tropism and autocrine of HGF/MET signaling. Compared with other orthotopic SCLC models, our model has a superior ability to form distant metastases. Therefore, our model will provide a valuable tool for the study of SCLC metastasis.

Intervenolin, a new antitumor compound with anti-Helicobacter pylori activity, from Nocardia sp. ML96-86F2.

Because stromal cells can regulate the growth and metastasis of tumor cells, a compound that modulates the interaction between the stromal cells and the tumor cells can control the tumor progression. In the course of our screening for such a compound, we have isolated a new compound, intervenolin, from the culture broth of Nocardia sp. ML96-86F2. Intervenolin inhibits the growth of human gastric and colorectal cancer cell lines in the coculture with the respective organ-derived stromal cells more strongly than that of the cancer cells cultured alone. Intervenolin shows antitumor effect against a xenograft model of human colorectal cancer cells in vivo. Furthermore, intervenolin exerts selective anti-Helicobacter pylori effect.

NBRI17671, a new antitumor compound, produced by Acremonium sp. CR17671.

The interaction between the receptor for advanced glycation end-product (RAGE) and amphoterin has an important role in tumor growth and metastasis. Because the abrogation of the interaction results in the inhibition of the tumor growth and metastasis, we designed a screening system for an inhibitor of the interaction between RAGE and amphoterin. In the course of our screening of the inhibitor, we isolated a novel natural compound NBRI17671 (1) from the fermentation broth of Acremonium sp. CR17671. We also modified 1 into a more active NBRI17671al (2). Although 1 at 50 g ml(-1) weakly inhibited binding of various cells to amphoterin, 2 at 50 g ml(-1) inhibited it by >50% of control. Compound 2 effectively inhibited the tumor growth of glioma and lung tumor xenografts in mice at 25 mg kg(-1). Furthermore, 2 was found to downregulate mitogen-activated protein kinase (MAPK) activity in the tumor cells.

Rubratoxin A specifically and potently inhibits protein phosphatase 2A and suppresses cancer metastasis.

Although cytostatin analog protein phosphatase 2A (PP2A)-specific inhibitors are promising candidates of a new type of anticancer drug, their development has been hindered because of their liability. To find new classes of PP2A-specific inhibitors, we conducted a screening with microbial metabolites and found that rubratoxin A, a classical mycotoxin, is a highly specific and potent inhibitor of the enzyme. While rubratoxin A inhibits PP2A at Ki = 28.7 nm, it hardly inhibited any other phosphatases examined. Rubratoxin B, a close analog, also specifically but weakly inhibits PP2A at Ki = 3.1 microM. The inhibition of intracellular PP2A in cultured cells is obviously observed with 20 microM rubratoxin A treatment for 3 h, inducing the overphosphorylation in PP2A substrate proteins. Although rubratoxins and cytostatin differ in the apparent structures, these compounds share similarities in the structures in detail and PP2A-binding manners. Rubratoxin A showed higher suppression of tumor metastasis and reduction of the primary tumor volume than cytostatin in mouse experiments. As a successor of cytostatin analogs, rubratoxin A should be a good compound leading to the development of antitumor drugs targeting PP2A.

Phthoxazolin A inhibits prostate cancer growth by modulating tumor-stromal cell interactions.

Because stroma in tumor tissues can promote prostate cancer development, modulation of tumor-stromal cell interactions may represent an attractive new strategy for cancer treatment. Here, we report that phthoxazolin A and its analog inthomycin B inhibit the growth of human prostate cancer DU-145 cells by modulating tumor-stromal cell interactions. Using an in vitro coculture system, in which prostate cancer cell growth is upregulated by prostate stromal cells (PrSC), we found that phthoxazolin A and inthomycin B strongly inhibited the growth of DU-145 cells when in coculture with PrSC compared to DU-145 cells cultured alone. Although PrSC consist of both fibroblasts and myofibroblasts, phthoxazolin A and inthomycin B inhibited the expression of smooth muscle alpha-actin, a myofibroblast marker, without affecting vimentin and beta-actin expression. Because myofibroblasts secrete various factors that can promote tumor cell growth, we examined whether the inhibitory compounds affected the secretion of such factors from PrSC. Proteomic analysis and reverse transcription-polymerase chain reaction revealed that phthoxazolin A and inthomycin B inhibited the expression of several insulin-like growth factor binding proteins and insulin-like growth factor (IGF)-I by PrSC. Transforming growth factor-beta1 increased myofibroblast numbers and IGF-I levels in PrSC. Phthoxazolin A inhibited transforming growth factor-beta1 activity without altering phosphorylation of the downstream molecule smad2. Furthermore, conditioned medium from phthoxazolin A-treated PrSC failed to increase the phosphorylation of IGF-IR and Akt in DU-145 cells. Taken together, our results suggested that phthoxazolin A acts as a small-molecule modulator of tumor-stromal cell interactions that can indirectly suppress prostate cancer cell growth through inhibition of IGF-I production by PrSC.

Establishment of a highly tumorigenic LNCaP cell line having inflammatory cytokine resistance.

Human androgen-dependent prostate cancer LNCaP cells are low tumorigenic even in immunodeficient mice and were killed by the synergistic effect of inflammatory cytokines, IL-beta and IL-6. To establish a highly tumorigenic LNCaP cell line, we isolated the cytokine-resistant LNCaP-CR cell line and examined the phenotypes. The parental LNCaP cells were induced to commit apoptosis by the addition of IL-1beta and IL-6, but LNCaP-CR cells showed strong resistance against the cytokine action. However, LNCaP-CR cells did not exhibit any resistance to various antitumor drugs investigated. While LNCaP cells formed only palpable tumors in SCID mice, LNCaP-CR cells readily made tumors and their growth was significantly higher than that of LNCaP cells. Moreover, LNCaP tumor-bearing mice gained the weight gradually, but LNCaP-CR tumor-bearing mice significantly lost their body weight. LNCaP-CR cells still responded to androgen action and expressed AR, erbB2, IL-1R, IL-6R, gp130, STAT3, p21, Bcl-2 and caspase-3 as well as LNCaP cells. These results indicate that LNCaP-CR cell line is a new type of tumorigenic LNCaP cell lines and should be useful for identifying responsible genes of tumorigenicity, cytokine resistance, and also cachexia.

Hygrolidin induces p21 expression and abrogates cell cycle progression at G1 and S phases.

Hygrolidin family antibiotics showed selective cytotoxicity against both cyclin E- and cyclin A-overexpressing cells. Among them, hygrolidin was the most potent and inhibited growth of solid tumor-derived cell lines such as DLD-1 human colon cancer cells efficiently more than that of hematopoietic tumor cells and normal fibroblasts. FACS analysis revealed that hygrolidin increased cells in G1 and S phases in DLD-1 cells. While hygrolidin decreased amounts of cyclin-dependent kinase (cdk) 4, cyclin D, and cyclin B, it increased cyclin E and p21 levels. Hygrolidin-induced p21 bound to and inhibit cyclin A-cdk2 complex more strongly than cyclin E-cdk2 complex. Furthermore, hygrolidin was found to increase p21 mRNA in DLD-1 cells, but not in normal fibroblasts. Thus, hygrolidin inhibited tumor cell growth through induction of p21. In respect to p21 induction, inhibition of vacuolar-type (H+)-ATPase by hygrolidin was suggested to be involved.