Phospho-aspirin (MDC-22) inhibits breast cancer in preclinical animal models: an effect mediated by EGFR inhibition, p53 acetylation and oxidative stress.

BACKGROUND: The anticancer properties of aspirin are restricted by its gastrointestinal toxicity and its limited efficacy. Therefore, we synthesized phospho-aspirin (PA-2; MDC-22), a novel derivative of aspirin, and evaluated its chemotherapeutic and chemopreventive efficacy in preclinical models of triple negative breast cancer (TNBC). METHODS: Efficacy of PA-2 was evaluated in human breast cancer cells in vitro, and in orthotopic and subcutaneous TNBC xenografts in nude mice. Mechanistic studies were also carried out to elucidate the mechanism of action of PA-2. RESULTS: PA-2 inhibited the growth of TNBC cells in vitro more potently than aspirin. Treatment of established subcutaneous TNBC xenografts (MDA-MB-231 and BT-20) with PA-2 induced a strong growth inhibitory effect, resulting in tumor stasis (79% and 90% inhibition, respectively). PA-2, but not aspirin, significantly prevented the development of orthotopic MDA-MB-231 xenografts (62% inhibition). Mechanistically, PA-2: 1) inhibited the activation of epidermal growth factor receptor (EGFR) and suppressed its downstream signaling cascades, including PI3K/AKT/mTOR and STAT3; 2) induced acetylation of p53 at multiple lysine residues and enhanced its DNA binding activity, leading to cell cycle arrest; and 3) induced oxidative stress by suppressing the thioredoxin system, consequently inhibiting the activation of the redox sensitive transcription factor NF-κB. These molecular alterations were observed in vitro and in vivo, demonstrating their relevance to the anticancer effect of PA-2. CONCLUSIONS: Our findings demonstrate that PA-2 possesses potent chemotherapeutic efficacy against TNBC, and is also effective in its chemoprevention, warranting further evaluation as an anticancer agent.

The anticancer effect of phospho-tyrosol-indomethacin (MPI-621), a novel phosphoderivative of indomethacin: in vitro and in vivo studies.

We have synthesized a novel derivative of indomethacin, phospho-tyrosol-indomethacin (PTI; MPI-621), and evaluated its anticancer efficacy in vitro and in vivo. PTI inhibited the growth of human colon, breast and lung cancer cell lines 6-30-fold more potently than indomethacin. In vivo, in contrast to indomethacin that was unable to inhibit colon cancer xenograft growth, PTI inhibited the growth of colon (69% at 10mg/kg/day, P < 0.01) and lung (91% at 15mg/kg/day, P < 0.01) subcutaneous cancer xenografts in immunodeficient mice, suppressing cell proliferation by 33% and inducing apoptosis by 75% (P < 0.05, for both). Regarding its pharmacokinetics in mice, after a single intraperitoneal injection of PTI, its plasma levels reached the maximum concentration (Cmax = 46 µM) at 2h (Tmax) and became undetectable at 4h. Indomethacin is the major metabolite of PTI, with plasma Cmax = 378 µM and Tmax = 2.5h; it became undetectable 24h postadministration. The cellular uptake of PTI (50-200 µM) at 6h was about 200-fold greater than that of indomethacin. Regarding its safety, PTI had no significant genotoxicity, showed less gastrointestinal toxicity than indomethacin and presented no cardiac toxicity. Mechanistically, PTI suppressed prostaglandin E2 production in A549 human lung cancer cells and strongly inhibited nuclear factor-κB activation in A549 xenografts. These findings indicate that PTI merits further evaluation as an anticancer agent.

Pancreas-specific ablation of beta1 integrin induces tissue degeneration by disrupting acinar cell polarity.

BACKGROUND & AIMS: Integrin contact with basement membrane is a major determinant of epithelial cell polarity. beta1 integrin heterodimers are the primary receptors for basement membrane in pancreatic acinar cells, which function to synthesize and directionally secrete digestive enzymes into a central lumen. Aberrant acinar secretion and exposure of the parenchyma to digestive enzyme activity lead to organ damage and pancreatitis. METHODS: beta1 integrin conditional knockout mice were crossed to Ptf1a-Cre mice to ablate beta1 integrin in the pancreas. Histopathology of aged and cerulein-treated mice were assessed by histology and immunocytochemistry. Directional secretion was determined in vitro by FM1-43 loading with cerulein stimulation. RESULTS: Pancreas-specific ablation of beta1 integrin led to progressive organ degeneration, associated with focal acinar cell necrosis and ductal metaplasia along with widespread inflammation and collagen deposition. beta1 Integrin-null pancreata were highly susceptible to cerulein-induced acute pancreatitis, displaying an enhanced level of damage with no loss in regeneration. Degenerating beta1 integrin-null pancreata were marked by disruption of acinar cell polarity. Protein kinase C epsilon, normally localized apically, was found in the cytoplasm where it can lead to intracellular digestive enzyme activation. beta1 Integrin-null acinar cells displayed indiscriminate secretion to all membrane surfaces, consistent with an observed loss of basolateral membrane localization of Munc18c, which normally prevents basal secretion of digestive enzymes. CONCLUSIONS: Ablation of beta1 integrin induces organ atrophy by disrupting acinar cell polarity and exposing the pancreatic parenchyma to digestive enzymes.

Ptf1a binds to and activates area III, a highly conserved region of the Pdx1 promoter that mediates early pancreas-wide Pdx1 expression.

The critical pancreatic transcription factor Pdx1 is expressed throughout the pancreas early but enriched in insulin-producing beta cells postnatally. Previous studies showed that the 5' conserved promoter regions areas I and II (Pdx1(PB)) direct endocrine cell expression, while an adjacent region (Pdx1(XB)) containing conserved area III directs transient beta-cell expression. In this study, we used Cre-mediated lineage tracing to track cells that activated these regions. Pdx1(PB)Cre mediated only endocrine cell recombination, while Pdx1(XB)Cre directed broad and early recombination in the developing pancreas. Also, a reporter transgene containing areas I, II, and III was expressed throughout the embryonic day 10.5 (E10.5) pancreas and gradually became beta cell enriched, similar to endogenous Pdx1. These data suggested that sequences within area III mediate early pancreas-wide Pdx1 expression. Area III contains a binding site for PTF1, a transcription factor complex essential for pancreas development. This site contributed to area III-dependent reporter gene expression in the acinar AR42J cell line, while PTF1 specifically trans-activated area III-containing reporter expression in a nonpancreatic cell line. Importantly, Ptf1a occupied sequences spanning the endogenous PTF1 site in area III of E11.5 pancreatic buds. These data strongly suggest that PTF1 is an important early activator of Pdx1 in acinar and endocrine progenitor cells during pancreas development.

Protein kinase D1 has a key role in wound healing and skin carcinogenesis.

Protein kinase D (PKD) is a family of stress-responsive serine/threonine kinases implicated in the regulation of diverse cellular functions including cell growth, differentiation, apoptosis, and cell motility. Although all three isoforms are expressed in keratinocytes, their role in skin biology and pathology is poorly understood. We recently identified a critical role for PKD1 during reversal of keratinocyte differentiation in culture, suggesting a potential proproliferative role in epidermal adaptive responses. Here, we generated mice with targeted deletion of PKD1 in the epidermis to evaluate the significance of PKD1 in normal and hyperplastic conditions. These mice displayed a normal skin phenotype, indicating that PKD1 is dispensable for skin development and homeostasis. Upon wounding, however, PKD1-deficient mice exhibited delayed wound re-epithelialization correlated with a reduced proliferation and migration of keratinocytes at the wound edge. In addition, the hyperplastic and inflammatory responses to topical phorbol ester were significantly suppressed, suggesting involvement of PKD1 in tumor promotion. Consistently, when subjected to the two-stage chemical skin carcinogenesis protocol, PKD1-deficient mice were resistant to papilloma formation when compared with control littermates. These results revealed a critical proproliferative role for PKD1 in epidermal adaptive responses, suggesting a potential therapeutic target in skin wound and cancer treatment.

A novel ibuprofen derivative with anti-lung cancer properties: synthesis, formulation, pharmacokinetic and efficacy studies.

Phospho-non-steroidal anti-inflammatory drugs (phospho-NSAIDs) are a novel class of NSAID derivatives with potent antitumor activity. However, phospho-NSAIDs have limited stability in vivo due to their rapid hydrolysis by carboxylesterases at their carboxylic ester link. Here, we synthesized phospho-ibuprofen amide (PIA), a metabolically stable analog of phospho-ibuprofen, formulated it in nanocarriers, and evaluated its pharmacokinetics and anticancer efficacy in pre-clinical models of human lung cancer. PIA was 10-fold more potent than ibuprofen in suppressing the growth of human non-small-cell lung cancer (NSCLC) cell lines, an effect mediated by favorably altering cytokinetics and inducing oxidative stress. Pharmacokinetic studies in rats revealed that liposome-encapsulated PIA exhibited remarkable resistance to hydrolysis by carboxylesterases, remaining largely intact in the systemic circulation, and demonstrated selective distribution to the lungs. The antitumor activity of liposomal PIA was evaluated in a metastatic model of human NSCLC in mice. Liposomal PIA strongly inhibited lung tumorigenesis (>95%) and was significantly (p<0.05) more efficacious than ibuprofen. We observed a significant induction of urinary 8-iso-prostaglandin F2αin vivo, which indicates that ROS stress probably plays an important role in mediating the antitumor efficacy of PIA. Our findings suggest that liposomal PIA is a potent agent in the treatment of lung cancer and merits further evaluation.

Aerosol administration of phospho-sulindac inhibits lung tumorigenesis.

Phospho-sulindac is a sulindac derivative with promising anticancer activity in lung cancer, but its limited metabolic stability presents a major challenge for systemic therapy. We reasoned that inhalation delivery of phospho-sulindac might overcome first-pass metabolism and produce high levels of intact drug in lung tumors. Here, we developed a system for aerosolization of phospho-sulindac and evaluated the antitumor efficacy of inhaled phospho-sulindac in an orthotopic model of human non-small cell lung cancer (A549 cells). We found that administration by inhalation delivered high levels of phospho-sulindac to the lungs and minimized its hydrolysis to less active metabolites. Consequently, inhaled phospho-sulindac (6.5 mg/kg) was highly effective in inhibiting lung tumorigenesis (75%; P < 0.01) and significantly improved the survival of mice bearing orthotopic A549 xenografts. Mechanistically, phospho-sulindac suppressed lung tumorigenesis by (i) inhibiting EGF receptor (EGFR) activation, leading to profound inhibition of Raf/MEK/ERK and PI3K/AKT/mTOR survival cascades; (ii) inducing oxidative stress, which provokes the collapse of mitochondrial membrane potential and mitochondria-dependent cell death; and (iii) inducing autophagic cell death. Our data establish that inhalation delivery of phospho-sulindac is an efficacious approach to the control of lung cancer, which merits further evaluation.

A novel Ras inhibitor (MDC-1016) reduces human pancreatic tumor growth in mice.

Pancreatic cancer has one of the poorest prognoses among all cancers partly because of its persistent resistance to chemotherapy. The currently limited treatment options for pancreatic cancer underscore the need for more efficient agents. Because activating Kras mutations initiate and maintain pancreatic cancer, inhibition of this pathway should have a major therapeutic impact. We synthesized phospho-farnesylthiosalicylic acid (PFTS; MDC-1016) and evaluated its efficacy, safety, and metabolism in preclinical models of pancreatic cancer. PFTS inhibited the growth of human pancreatic cancer cells in culture in a concentration- and time-dependent manner. In an MIA PaCa-2 xenograft mouse model, PFTS at a dose of 50 and 100 mg/kg significantly reduced tumor growth by 62% and 65% (P < .05 vs vehicle control). Furthermore, PFTS prevented pancreatitis-accelerated acinar-to-ductal metaplasia in mice with activated Kras. PFTS appeared to be safe, with the animals showing no signs of toxicity during treatment. Following oral administration, PFTS was rapidly absorbed, metabolized to FTS and FTS glucuronide, and distributed through the blood to body organs. Mechanistically, PFTS inhibited Ras-GTP, the active form of Ras, both in vitro and in vivo, leading to the inhibition of downstream effector pathways c-RAF/mitogen-activated protein-extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK1/2 kinase and phosphatidylinositol 3-kinase/AKT. In addition, PFTS proved to be a strong combination partner with phospho-valproic acid, a novel signal transducer and activator of transcription 3 (STAT3) inhibitor, displaying synergy in the inhibition of pancreatic cancer growth. In conclusion, PFTS, a direct Ras inhibitor, is an efficacious agent for the treatment of pancreatic cancer in preclinical models, deserving further evaluation.

Targeting mitochondrial STAT3 with the novel phospho-valproic acid (MDC-1112) inhibits pancreatic cancer growth in mice.

New agents are needed to treat pancreatic cancer, one of the most lethal human malignancies. We synthesized phospho-valproic acid, a novel valproic acid derivative, (P-V; MDC-1112) and evaluated its efficacy in the control of pancreatic cancer. P-V inhibited the growth of human pancreatic cancer xenografts in mice by 60%-97%, and 100% when combined with cimetidine. The dominant molecular target of P-V was STAT3. P-V inhibited the phosphorylation of JAK2 and Src, and the Hsp90-STAT3 association, suppressing the activating phosphorylation of STAT3, which in turn reduced the expression of STAT3-dependent proteins Bcl-xL, Mcl-1 and survivin. P-V also reduced STAT3 levels in the mitochondria by preventing its translocation from the cytosol, and enhanced the mitochondrial levels of reactive oxygen species, which triggered apoptosis. Inhibition of mitochondrial STAT3 by P-V was required for its anticancer effect; mitochondrial STAT3 overexpression rescued animals from the tumor growth inhibition by P-V. Our results indicate that P-V is a promising candidate drug against pancreatic cancer and establish mitochondrial STAT3 as its key molecular target.