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Purpose The development of drug-resistant phenotypes has been a major obstacle to Cisplatin (CDDP) use in non-small cell lung cancer (NSCLC). We aimed to identify some of the molecular mechanisms that underlie CDDP resistance by using microarray expression analysis. Methods and Materials H460 cells were treated with cisplatin. Differences between cisplatin-resistant lung cancer cells (CDDP-R) and parental H460 cells were studied using Western blot, MTS and clongenic assays, in vivo tumor implantation, and microarray analysis. CDDP-R cells were treated with human recombinant insulin-like growth factor binding protein-3 (IGFBP-3) and siRNA targeting insulin-like growth factor-1 receptor (IGF-1R). Results CDDP-R cells illustrated greater expression of the markers CD133 and ALDH, more rapid in vivo tumor growth, more resistance to cisplatin- and etoposide-induced apoptosis, and greater survival after treatment with cisplatin or radiation than the parental H460 cells. Also, CDDP-R demonstrated decreased expression of IGFBP-3 and increased activation of IGF-1R signaling as compared to parental H460 cells in the presence of IGF-1. Human recombinant IGFBP-3 reversed cisplatin resistance in CDDP-R cells, and targeting of IGF-1R using siRNA resulted in sensitization of CDDP-R-cells to cisplatin and radiation. Conclusions The IGF-1 signaling pathway contributes to CDDP-R resistance to cisplatin and radiation. Thus, this pathway represents a potential target for improved lung cancer response to treatment.

Introduction Defects in the apoptosis pathway limit the effectiveness of radiation in non-small cell lung cancer (NSCLC) therapy. BV6 is an antagonist of cIAP1 and XIAP, members of the inhibitors of apoptosis (IAP) family. We investigated the potential of BV6 to sensitize NSCLC cell lines to radiation. Methods HCC193 and H460 lung cancer cell lines were treated with BV6 to investigate the effects of drug administration on cell proliferation, apoptosis, inhibition of XIAP and cIAP1, and radiosensitivity. Subsequent immunoblotting and Hoechst staining were utilized to determine the role of apoptosis in radiosensitization. Finally, the pathway of apoptosis was characterized by western blot analysis for cleaved caspase-8 and cleaved caspase-9, and ELISA assays for TNF-α. Results HCC193 was found to be more sensitive than H460 to BV6-induced apoptosis in a concentration-dependent and time-dependent manner. BV6 significantly sensitized both cell lines to radiation (HCC193—DER=1.38, p<0.05 at 1μM BV6; H460—DER=1.42, p<0.05 at 5μM BV6), but a higher concentration of and longer incubation time with BV6 was necessary for H460 cells. The BV6-induced radiosensitization of HCC193 favored the extrinsic pathway of apoptosis, while that of H460 favored the intrinsic pathway. Conclusions BV6, an IAP antagonist, significantly enhanced the radiosensitization of HCC193 and H460 cells in vitro. More research is warranted to test the mechanism of action of BV6, and to assess its potential in vivo and in the clinical setting.

Crizotinib (PF02341066) is a tyrosine kinase inhibitor of ALK that has been shown to selectively inhibit growth of cancer cells that harbor the EML4-ALK fusion, found in a subset of patients with non-small cell lung cancer (NSCLC). While in clinical trials PF02341066 has shown a significant therapeutic benefit as a single agent, the effectiveness of combining it with other therapeutic modalities including ionizing radiation remains unknown. To further elucidate the role of PF02341066 in tumor inhibition, we examined its effects alone and in combination with radiation on downstream signaling, apoptosis, and radiosensitivity in two NSCLC cell lines in vitro: H3122, which harbors the EML4-ALK fusion; and H460, which does not. We also examined the in vivo effects of PF02341066 in H3122 mouse xenografts. In the H3122 cell line, PF02341066 inhibited phosphorylation of ALK and its downstream effectors: AKT, ERK, and STAT3. H3122 cells treated with a combination of PF02341066 and radiation showed an increase in cellular apoptosis and were sensitized to radiation therapy (dose enhancement ratio, 1.43; p < 0.0001). Moreover, in a H3122 xenograft model, the combined treatment resulted in greater tumor growth inhibition than either treatment alone (p < 0.05). None of these effects was observed in the EML4-ALK-negative H460 cells. Our findings indicate that PF02341066 acts as a radiation sensitizer in cells harboring the EML4-ALK fusion, providing a rationale for a clinical trial combining ALK inhibitor with radiation in the NSCLC expressing ALK.

Introduction Persistent STAT3 activation contributes to lung carcinogenesis. Survivin, one of STAT3-regulated genes, is antiapoptotic and confers cancer radioresistance. Methods We tested whether TG101209, a small-molecule inhibitor of JAK2 (a STAT3-activating tyrosine kinase), affected survivin expression and sensitized lung cancer to radiation. We investigated whether inhibition of JAK2 signaling with TG101209 can be used to reduce survivin expression and enhance radiosensitivity of lung cancer cells in vitro and tumor growth delay in vivo. JAK2 downstream signaling, including PI3-K/Akt and Ras/MAPK/ERK pathways, was also explored. Results TG101209 inhibited STAT3 activation and survivin expression, and sensitized HCC2429 (DER=1.34,p=0.002) and H460 (DER=1.09,p=0.006) cells to radiation in clonogenic assays. Radiation promoted phospho-Akt and phospho-ERK in H460 cells, while their levels were unchanged in HCC2429. After treatment with TG101209, phospho-ERK protein levels were reduced in both HCC2429 and H460 cells. HCC2429 cells transfected with K-Ras-12V mutant were more resistant to radiation- and TG101209-induced apoptosis than wild-type control cells. In vivo, addition of TG101209 to radiation in lung xenografts produced a significant tumor growth delay (>10 days) compared to radiation alone and was well tolerated. Immunohistochemistry staining of tumor sections showed that TG101209 increased apoptosis and decreased cell proliferation and vascular density, suggesting that TG101209 also has antiangiogenic effects. Conclusions TG101209 enhanced the effects of radiation in lung cancer in vitro and in vivo. This study suggests the potential utility of selecting lung cancer patients according to K-Ras mutation status for future clinical trials testing combination of TG101209 and radiotherapy.

Burn induces myeloid-derived suppressor cells (MDSCs), a heterogeneous population of immature polymorphonuclear neutrophils (PMNs) and monocytes, which protect against infection. Previous work from our laboratory demonstrated that inflammatory monocytes (iMos) were the major MDSC source of TNF-α in the postburn spleen, and we hypothesized that they were also the major source of postburn IL-10. To test this hypothesis, we examined cytokine production by postburn CCR2 knockout (KO) mice, which have fewer iMos than burn wild-type (WT) splenocytes, but equal numbers of PMNs and F4/80 macrophages. Using cell sorting and/or intracellular cytokine techniques, we examined IL-10 production by postburn PMNs and iMos. Finally, we compared IL-10 production by postburn PMNs and iMos with culture-derived MDSCs. Splenocytes from postburn CCR2 KO mice produced less IL-6 and TNF-α than WT burn splenocytes in response to LPS, but KO and WT burn splenocytes produced equal amounts of IL-10 in response to peptidoglycan. Depletion of PMNs from postburn splenocytes led to reductions in IL-10 and increases in IL-6 and TNF-α in response to peptidoglycan, but not in response to LPS. Sorting or intracellular cytokine techniques gave consistent results: Burn PMNs made more IL-10 than sham PMNs and also more IL-10 than burn or sham iMos. Polymorphonuclear neutrophil and iMos subpopulations from culture-derived MDSCs produced the same cytokine profiles in response to LPS and peptidoglycan as did the PMNs and iMos from postburn spleens: PMNs made IL-10, whereas iMos made IL-6. Finally, LPS-induced mortality of burn mice was made worse by anti-Gr-1 depletion of all PMNs and 66% of iMos from burn mice. This suggests that PMNs play a primarily anti-inflammatory role in vitro and in vivo.

A patient with newly diagnosed right lung cancer had transient 18 F-fluorodeoxyglucose (FDG)-avid left axillary lymph nodes and intense splenic FDG uptake on positron emission tomography (PET)/computed tomography (CT). History revealed that the patient received a leftsided influenza vaccine 2-3 days before the examination. Although inflammatory FDG uptake in ipsilateral axillary nodes is reported, to our knowledge, this is the first report of visualization of the systemic immune response in the spleen related to the influenza vaccination on FDG-PET/ CT. The history, splenic uptake and time course on serial FDG-PET/CT helped to avoid a false-positive interpretation for progressing lung cancer and alteration of the radiation therapy plan.

Introduction Survivin, an inhibitor of apoptosis protein (IAP) and key regulator of mitosis, is up-regulated in a variety of cancers and is often associated with a worse prognosis. Terameprocol down-regulates the Sp1-mediated transcription of survivin and Cdk1, which is important for cell cycle progression, as well as many other proteins. Survivin inhibition has previously been shown to result in the induction of apoptosis and radiosensitization. Methods This study examined the effects of terameprocol administration on survivin transcription and expression in HCC2429 and H460 lung cancer cells. We also examined the combined effects of radiation and terameprocol on apoptosis and radiosensitivity. Results Using immunoblot analysis and luciferase assays, we confirmed that terameprocol decreases survivin transcription and protein expression. Ultimately, however, decreases in survivin expression failed to correlate with an increase in apoptosis. Nonetheless, clonogenic assay revealed that terameprocol induces increased radiosensitization in HCC2429 (DER = 1.26, p = 0.019) and H460 (DER = 1.18, p = 0.001) cells. Additionally, the data show no effect of terameprocol on cell cycle in either HCC2429 or H460 cells. Conclusions Terameprocol significantly enhances the sensitivity of non-small cell lung carcinoma cell lines to radiation therapy, although the mechanism of action remains unclear. Further study is warranted to assess the potential of terameprocol as an agent that may enhance the therapeutic ratio of radiotherapy in lung cancer.