Parathyroid hormone-related protein (PTHrP) is expressed in more advanced, aggressive tumors and may play an active role in cancer progression. This study investigated the effects of PTHrP on apoptosis after UV irradiation, Fas ligation, or staurosporine treatment in BEN human squamous lung carcinoma cells. Cells at 70% confluency were treated for 24 h with 100 nM PTHrP-(1-34), PTHrP-(38-64), PTHrP-(67-86), PTHrP-(107-139), or PTHrP-(140-173) in media with serum, exposed for 30 min to UV-B radiation (0.9 mJ/cm2), and maintained for another 24 h. Caspase-3, caspase-8, and caspase-9 activities increased fivefold. Pretreatment with PTHrP-(1-34) and PTHrP-(140-173) ameliorated apoptosis after UV irradiation, as indicated by reduced caspase activities, increased cell protein, decreased nuclear condensation, and increased clonal survival. Other peptides had no effect on measures of apoptosis. PTHrP-(140-173) also reduced caspase activities after Fas ligation by activating antibody, but neither peptide had effects on caspase-3 or caspase-9 activity after 1 μM staurosporine. These data indicate that PTHrP-(1-34) and PTHrP-(140-173) protect against death receptor-induced apoptosis in BEN lung cancer cells but are ineffective against mitochondrial pathways. PTHrP contributes to lung cancer cell survival in culture and could promote cancer progression in vivo. The mechanism for the protective effect against apoptosis remains to be determined.
Parathyroid hormone-related protein (PTHrP)-(1–34) and PTHrP-(140–173) protect lung cancer cells from apoptosis after ultraviolet (UV) irradiation. This study evaluated upstream signaling in PTHrP-mediated alteration of lung cancer cell sensitivity to apoptosis. The two peptides increased cAMP levels in BEN lung cancer cells by 15–35% in a dose-dependent fashion, suggesting signaling through protein kinase A (PKA). In line with this view, the PKA inhibitor H89 abrogated the protective effects of PTHrP-(1–34) and PTHrP-(140–173) against caspase activation and DNA loss. PKA activation by forskolin, 3-isobutyl-1-methylxanthine (IBMX), or 8-(4-chlorophenylthio)adenosine 3′,5′-cyclic monophosphate attenuated and H89 augmented apoptosis after UV exposure as indicated by caspase-3 activation, cell DNA loss, and morphological criteria. Studies with IBMX and varying doses of forskolin indicated that small increases in cAMP, on the order of those generated by IBMX alone and the PTHrP peptides, were sufficient to protect lung cancer cells from apoptosis. In summary, PTHrP-(1–34) and PTHrP-(140–173) stimulate PKA in lung carcinoma cells and protect cells against UV-induced caspase-3 activation and DNA fragmentation. PKA activation by other means also induces resistance to apoptosis, and the protective effect of the PTHrP peptide is blocked by PKA inhibition. Thus PKA appears to have a role in the regulatory effects of PTHrP on lung cancer cell survival.
Hyaluronan (HA) is a large glycosaminoglycan, and a significant component of stromal extracellular matrix (ECM) in many tissues and malignancies. It's accumulation on cancer cells and the surrounding stroma predicts unfavorable disease outcome, suggesting that HA enhances tumor growth and progression. PEGPH20 is a pegylated recombinant human hyaluronidase that depletes HA substrate from extracellular tumor microenvironment, decreases tumor interstitial fluid pressure (IFP) and water content and increases tumor vascular area. We have previously demonstrated significant anti-tumor effects of PEGPH20 when used as a single agent and in combination with Docetaxel and liposomal Doxorubicin in peritibial PC3 xenograft prostate carcinoma model. Herein, we evaluated PEGPH20 therapeutic potential in combination with Gemcitabine in the HA-high (BxPC-3) and HA-low (AsPC-1, MIAPaCa-2) subcutaneous pancreatic cancer xenograft models. Assessment of HA levels in the culture supernatants of BxPC-3, AsPC-1 and MIAPaCa-2 cell lines produced 9.4, 1.2 and 0.2 µg of HA per 1E6 tumor cells, respectively following 3 days of cell culture incubation. These HA levels correlated with the respective in vivo HA tumor expression and in vitro area of fixed red blood cell exclusion (‘halo’) for each of the three tumor lines. In the BxPC-3 model IV administration of PEGPH20 at 4.5 mg/kg concurrently with Gemcitabine given IP at 30 mg/kg on Days 1, 4, 7, 10 followed by second treatment cycle on Days 22, 25, 28 and 31 resulted in a synergistic antitumor effect with a significantly superior tumor growth inhibition (TGI) compared to Gemcitabine alone. Following completion of the 1st treatment cycle, PEGPH20 + Gemcitabine (4.5 mg/kg + 30 mg/kg) induced 51% TGI, compared to 29% TGI for PEGPH20 (4.5 mg/kg) and 13% TGI for Gemcitabine (30 mg/kg) by Day 21 post-treatment. Initiation of the 2nd treatment cycle with PEGPH20 + Gemcitabine (4.5 mg/kg + 30 mg/kg) at Day 22 resulted in potent and sustained TGI of large size (>1,000 mm3) tumors with tumor volumes remaining relatively unchanged until study termination (Day 36). PEGPH20 activity in the sera of BxPC-3 tumor bearing mice was found to be elevated following treatment with either PEGPH20 alone or PEGPH20 + Gemcitabine; whereas measurements of soluble HA in the same serum samples were below quantification levels. In contrast to the observed PEGPH20 + Gemcitabine induction of synergistic anti-tumor activity in the HA-high BxPC-3 tumor, no significant anti-tumor effects were evident following treatment of HA-low AsPC-1 and MIAPaCa-2 tumors with identical regimens and dosages. We conclude that PEGPH20 could represent an innovative potential treatment approach that may provide improved therapeutic benefit and survival when used with current standard of care treatments for highly HA positive pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5392.
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