NFκB and TNFα as individual key molecules associated with the cisplatin-resistance and radioresistance of lung cancer.

Cisplatin-resistant (A549CisR and H292CisR) and radioresistant (A549R26 and H292R22) sub-line non-small cell lung cancer (NSCLC) cells were developed in our lab by long term treatment of parental cells with cisplatin or radiation. Our data showed no cross-resistance between these two sets of cell lines, indicating that molecular mechanisms of developing each resistance may be different. Using these sub-line cells, we sought to reveal the most significantly up-regulated molecules in cisplatin-resistant and radioresistant lung cancer cells, compared with parental cells. In qPCR analyses of screening DNA repair and cell survival-associated molecules, we identified NFκB and TNFα as the most significantly up-regulated molecules in cisplatin-resistant and radioresistant lung cancer cells, respectively, compared with parental cells. Western blot analysis of parental vs. resistant cells and the IHC staining of tumor tissues of A549P, A549CisR, and A549R26 cell-derived xenografts in mice confirmed such results. Next, studies using specific inhibitors of NFκB and TNFα and experiments using NFκB and TNFα-knocked down cells showed that inhibition or knockdown of NFκB overcame cisplatin-resistance, while inhibition or knockdown of TNFα increased radiosensitivity of radioresistant lung cancer cells. Therefore, these two molecules may be used as markers of the prognosis/diagnosis of individual resistance development during lung cancer treatment.

Glucocorticoid receptor upregulation increases radioresistance and triggers androgen independence of prostate cancer.

BACKGROUND: Despite the overall success of radiotherapy, a significant number of patients develop radioresistance, which leads to local regional recurrence and distant metastasis. We studied whether repeated radiation treatment promotes androgen-independent survival of prostate cancer (PCa) cells and their metastatic potential. We also studied whether glucocorticoid receptor (GR) increase in radioresistant cells is associated with acquisition of these aggressive characteristics. METHODS: Radioresistant LNCaP (LNCaPR18) and C4-2 (C4-2R26) PCa sublines were developed by repeated radiation treatments of parental cells. Levels and activations of androgen receptor (AR) and GR in radioresistant PCa cells and respective parental cells were investigated in quantitative real-time polymerase chain reaction/Western blot analyses and immunofluorescence staining. Androgen-independent survival of radioresistant cells was tested in in vitro cell growth assays and the castration-resistant survival of these cell-derived tumors were investigated in mouse xenografts. RESULTS: Higher GR levels, but lower AR levels were detected in radioresistant cells than in parental cells. Radiation-induced GR upregulation was associated with increased intracellular cyclic adenosine monophosphate. As a consequence of GR activation, LNCaPR18 cells survived well in an androgen-depleted culture condition while parental cells could not. Results of in vivo mouse studies showed survival of LNCaPR18 cell-derived tumors in castrated mice while parental cell-derived tumors regressed. The growth of LNCaPR18 cell-derived tumors in castrated mice was impaired when treated with the anti-GR agent mifepristone. In experiments with C4-2/C4-2R26 cell sets, GR activation in C4-2R26 cells increased their metastatic potential. CONCLUSION: GR activation in radioresistant cells mediates androgen independence and facilitates PCa progression.

In vitro-induced M2 type macrophages induces the resistance of prostate cancer cells to cytotoxic action of NK cells.

Previous reports, including our experimental results, showed that macrophages migrate to prostate cancer (PCa) cells. We tested whether the migrated macrophages affect the susceptibility of castration-resistant PCa (CRPC) cells to cytotoxic actions of natural killer (NK) cells. We found treatment of tumor cells with the conditioned media (CM) of the PMA/IL-4 treated THP-1 cells (M2 type macrophages) (THP-1 CM) decreased the susceptibility of tumor cells to NK cell cytotoxicity, as a result of increased programmed death receptor ligand 1 (PD-L1) and decreased NK group 2D (NKG2D) ligands in CRPC cells. Meanwhile, the decreased susceptibility of tumor cells was also detected when NK cells were treated with THP-1 CM and used in NK cell cytotoxicity tests. Therefore, we observed higher resistance of CRPC cells when both tumor and NK cells were treated with THP-1 CM than when tumor cells or NK cells were individually treated. We further discovered that the PMA/IL-4 treated THP-1 cells secrete a high level of IL-6, so blocking the IL-6 action significantly decreased the PD-L1 level while recovering the NKG2D ligands, thus increasing the susceptibility of CRPC cells to NK cell action. Moreover, we discovered that JAK-Stat3 is the most critical IL-6 downstream signaling in triggering the THP-1 CM effect. Consequently, we found the susceptibility of CRPC cells to NK cells was increased when either JAK or Stat 3 inhibitor was added when tumor cells were treated with THP-1 CM, and that the best effect was observed when the JAK inhibitor and PD-L1 Ab were added together.

Adipocytes affect castration-resistant prostate cancer cells to develop the resistance to cytotoxic action of NK cells with alterations of PD-L1/NKG2D ligand levels in tumor cells.

BACKGROUND: Obesity affects prostate cancer (PCa) progression, and the periprostatic adipose tissue adjacent to the prostate is considered a driving force of disease progression. Adipocytes are the main cell population in adipose tissues and their paracrine role contributes to PCa progression, however its implication in modulating immune reactions remains largely unknown. We investigated the adipocyte role in controlling the susceptibility of castration-resistant PCa (CRPC) cells to the cytotoxic action of natural killer (NK) cells. METHODS: Using primary NK cells as the NK cell source, NK cell cytotoxicities to CRPC cells, either control media treated or adipocyte-conditioned media (CM) treated, were tested in lactate dehydrogenase (LDH) release-based assays. The levels of programmed death receptor ligand (PD-L1) and NK group 2D (NKG2D) ligands in adipocyte CM-treated CRPC cells were analyzed in qPCR analyses. Effects of blocking adipocyte action on altering PD-L1/NKG2D ligand levels and the susceptibility of CRPC cells to NK cell cytotoxicity were investigated. RESULTS: We found NK cell cytotoxicity to CRPC cells decreases when tumor cells are treated with adipocyte CM associated with PD-L1 and NKG2D ligand level alterations. Further, we discovered that the JAK/Stat3 signaling pathway was responsible for the adipocyte CM effect. Two adipokine molecules, IL-6 and leptin, were shown to be important in activation of the JAK/Stat3 signaling in CRPC cells to modulate the PD-L1/NKG2D ligand level alteration. Adding the inhibitors of JAK/Stat3 signaling or neutralizing antibodies of IL-6 or leptin increased the susceptibility of CRPC cells to NK cell action. CONCLUSIONS: Blocking the adipocyte effect by inhibiting the IL-6/leptin-JAK/Stat3 signaling axis may enhance NK cell mediated immunity to CRPC cells and this strategy may help to develop future therapeutics to treat obese PCa patients.

FASN-TGF-ß1-PD-L1 axis contributes to the development of resistance to NK cell cytotoxicity of cisplatin-resistant lung cancer cells.

Cisplatin remains the most effective therapy for non-small cell lung cancer (NSCLC). We previously have found cisplatin-resistant lung cancer cells (A549CisR and H157CisR) were more resistant to natural killer (NK) cell-mediated cytotoxicity than parental cells. We also discovered that fatty acid synthase (FASN) levels in cisplatin-resistant cells were significantly higher than in parental cells. To reveal whether a link exists between the up-regulated FASN levels and higher resistance to NK cell cytotoxicity, we performed inhibition studies using a FASN inhibitor and applied the FASN knockdown approach. In both approaches, we found that the FASN inhibition/knockdown significantly increased the susceptibility of cisplatin-resistant cells to NK cell cytotoxicity. We further found such decreased susceptibility was associated with an increased programmed death receptor ligand (PD-L1) level in cisplatin-resistant cells. In mechanisms studies, TGF-ß1 was found to be the FASN downstream signaling molecule that was responsible for modulating the PD-L1 levels in cisplatin-resistant cells. Accordingly, TGF-ß1 inhibition resulted in significantly increased susceptibility of cisplatin-resistant cells to NK cell cytotoxicity. We suggest that the inhibition of FASN-TGFß1-PD-L1 axis may improve the efficacy of immunotherapy in treating cisplatin-resistant lung cancer.

Cisplatin treatment increases stemness through upregulation of hypoxia-inducible factors by interleukin-6 in non-small cell lung cancer.

Cisplatin-resistant A549 and H157 (A549CisR and H157CisR) non-small cell lung cancer cells show increased stemness of cancer stem cells (CSCs) compared to their parental cells. We investigated whether interleukin-6 (IL-6) signaling contributes to this increased stemness in cisplatin-resistant cells. When A549CisR and H157CisR cells were treated with neutralizing IL-6 antibody, decreased cisplatin resistance was observed, whereas IL-6 treatment of parental cells resulted in increased cisplatin resistance. Expression of the CSC markers was significantly upregulated in IL-6-expressing scramble cells (in vitro) and scramble cell-derived tumor tissues (in vivo) after cisplatin treatment, but not in IL-6 knocked down (IL-6si) (in vitro) cells and in IL-6si cell-derived tumor tissues (in vivo), suggesting the importance of IL-6 signaling in triggering increased stemness during cisplatin resistance development. Hypoxia inducible factors (HIFs) were upregulated by IL-6 and responsible for the increased CSC stemness on cisplatin treatment. Mechanism dissection studies found that upregulation of HIFs by IL-6 was through transcriptional control and inhibition of HIF degradation. Treatment of HIF inhibitor (FM19G11) abolished the upregulation of CSC markers and increased sphere formations in IL-6 expressing cells on cisplatin treatment. In all, IL-6-mediated HIF upregulation is important in increasing stemness during cisplatin resistance development, and we suggest that the strategies of inhibiting IL-6 signaling or its downstream HIF molecules can be used as future therapeutic approaches to target CSCs after cisplatin treatment for lung cancer.

Neonatal oxygen increases sensitivity to influenza A virus infection in adult mice by suppressing epithelial expression of Ear1.

Oxygen exposure in premature infants is a major risk factor for bronchopulmonary dysplasia and can impair the host response to respiratory viral infections later in life. Similarly, adult mice exposed to hyperoxia as neonates display alveolar simplification associated with a reduced number of alveolar epithelial type II cells and exhibit persistent inflammation, fibrosis, and mortality when infected with influenza A virus. Because type II cells participate in innate immunity and alveolar repair, their loss may contribute to oxygen-mediated sensitivity to viral infection. A genomewide screening of type II cells identified eosinophil-associated RNase 1 (Ear1). Ear1 was also detected in airway epithelium and was reduced in lungs of mice exposed to neonatal hyperoxia. Electroporation-mediated gene delivery of Ear1 to the lung before infection successfully reduced viral replication and leukocyte recruitment during infection. It also diminished the enhanced morbidity and mortality attributed to neonatal hyperoxia. These findings demonstrate that novel epithelial expression of Ear1 functions to limit influenza A virus infection, and its loss contributes to oxygen-associated epithelial injury and fibrosis after infection. People born prematurely may have defects in epithelial innate immunity that increase their risk for respiratory viral infections.

Antioxidant properties of quercetin.

UNLABELLED: Quercetin, a plant-derived aglycone form of flavonoid glycosides, has been used as a nutritional supplement and may be beneficial against a variety of diseases, including cancer. We examined the antioxidant properties of quercetin. The reduction potential of quercetin was measured at various pH values using voltammetric methods, and its total antioxidant capacity (TAC) was measured using the phosphomolybdenum method. The effect of quercetin on production of reactive oxygen species (ROS) and nitric oxide (NO) in LPS-stimulated human THP-1 acute monocytic leukemia cells was determined by flow cytometry using CM-H2DCFDA dye. The results were compared with curcumin, a natural product exhibiting a similar range of reported health benefits. RESULTS: 1) Quercetin has a higher reduction potential compared with curcumin at three different pH settings and is comparable to Trolox at pH 7-9.5; 2) its TAC is 3.5 fold higher than curcumin; 3) it reduced LPS-induced ROS to near normal levels; 4) it reduced LPS-induced NO production. These data provide a physico-chemical basis for comparing antioxidants, with potential benefits individually or in combination.

[Corrigendum] Increased infiltration of macrophages to radioresistant lung cancer cells contributes to the development of the additional resistance of tumor cells to the cytotoxic effects of NK cells.

Subsequently to the publication of the above paper, the authors have realized that the western blots featured in Fig. 5B were inadvertently copied across from Fig. 4B owing to an error made during the figure compilation process. The corrected version of Fig. 5 is featured on the next page, showing the correct data for the western blot analysis of the programmed death receptor ligand 1 level in radioresistant lung cancer cells under the specified experimental conditions. Note that these changes do not affect the interpretation of the data or the conclusions reported in this paper, and all the authors agree to this correction. The authors apologize to the Editor and to the readership of the Journal for any inconvenience caused. [the original article was published in International Journal of Oncology 53: 317-328, 2018; DOI: 10.3892/ijo.2018.4394].

Involvement of a p53-independent and post-transcriptional up-regulation for p21WAF/CIP1 following destabilization of the actin cytoskeleton.

The tumor suppressor p21WAF/CIP1 mediates the proliferation arrest via p53-dependent or -independent gene transactivation following distinct environmental stresses. In this study, we show that direct destabilization of the actin cytoskeleton by actin-targeting reagents leads to a p53-independent up-regulation of p21WAF/CIP1. The actin-targeting agent cytochalasin B (10 microM) quickly disrupted the actin cytoskeleton of p53 wild-type and p53-null cells accompanied by up-regulation of p21WAF/CIP1. Nevertheless, both total p53 and ser-15 phosphorylated p53 were not accumulated concomitantly, compared to the effect caused by ionizing irradiation. P53-independent up-regulation of p21WAF/CIP1 was also observed by two other actin-targeting agents cytochalasin D and latrunculin B, but not by the microtubule inhibitor colcemid. Furthermore, we showed that p21WAF/CIP1 mRNA level was not increased, whereas the protein degradation was delayed. A reduction of ubiquitination for p21WAF/CIP1 protein was detected using immunoprecipitation/immunoblot analysis. Up-regulation of p21WAF/CIP1 was not associated with cytotoxicity induced by cytochalasin B that influenced DNA integrity and plating efficiency only after 24 h of treatment. In addition, up-regulated p21WAF/CIP1 was accompanied by reduction of phosphorylation on retinoblastoma (Rb) protein in p53-null cells, implying that p21WAF/CIP1 might in part account for the molecular regulation of cytochalasin B induced G1 phase arrest. Together, current results suggest that p21WAF/CIP1 level can be mediated by actin organization in the absence of p53 via a post-transcriptional machinery, and it may contribute to the growth ablation by agents targeting the actin cytoskeleton.

Replication of murine mitochondrial DNA following irradiation.

The effect of radiation on the mitochondrial genome in vivo is largely unknown. Though mitochondrial DNA (mtDNA) is vital for cellular survival and proliferation, it has little DNA repair machinery compared with nuclear DNA (nDNA). A better understanding of how radiation affects mtDNA should lead to new approaches for radiation protection. We have developed a new system using real-time PCR that sensitively detects the change in copy number of mtDNA compared with nDNA. In each sample, the DNA sequence coding 18S rRNA served as the nDNA reference in a run simultaneously with a mtDNA sequence. Small bowel collected 24 hours after 2 Gy or 4 Gy total body irradiation (TBI) exhibited increased levels of mtDNA compared with control mice. A 4 Gy dose produced a greater effect than 2 Gy. Similarly, in bone marrow collected 24 hours after 4 Gy or 7 Gy TBI, 7 Gy produced a greater response than 4 Gy. As a function of time, a greater effect was seen at 48 hours compared with 24 hours. In conclusion, we found that radiation increased the ratio of mtDNA:nDNA and that this effect seems to be tissue independent and seems to increase with radiation dose and duration following radiation exposure.

Preclinical study using androgen receptor (AR) degradation enhancer to increase radiotherapy efficacy via targeting radiation-increased AR to better suppress prostate cancer progression.

BACKGROUND: While androgen deprivation therapy (ADT) and radiotherapy (RT) are currently used together to treat locally advanced prostate cancer (PCa), RT might have the adverse effect of increasing the PCa androgen receptor (AR) protein expression, which might then increase the resistance to continued RT. METHODS: We used multiple assays for RT sensitivity, protein and RNA expression of AR and related DDR genes, ROS level, DNA damage/repair level, cell cycle and apoptosis. All statistical comparisons were analyzed with t-test or one-way ANOVA. FINDINGS: We demonstrated that RT induced AR expression in C4-2 and CWR22Rv-1 cells. We found that combining RT and ASC-J9®, but not the antiandrogen, Enzalutamide, could increase radiosensitivity via inducing DNA damage, altering the AR mediated and DNA repair pathways, and activating apoptosis. ASC-J9® had little effects on normal bladder cells. INTERPRETATION: Targeting ionizing radiation (IR)-increased AR with the AR degradation enhancer, ASC-J9®, could increase the radiosensitivity while sparing adjacent normal tissue. Mechanism dissection revealed that ASC-J9®, but not Enzalutamide, treatment could increase radiosensitivity via inducing DNA damage, altering DNA repair pathways, as well as activating the IR-induced apoptosis via suppressing the pATR-CHK1 signals. Importantly, results from preclinical studies using an in vivo mouse model also demonstrated that combining RT with ASC-J9® to target AR led to better therapeutic efficacy to suppress PCa progression.

IL-6 signaling contributes to radioresistance of prostate cancer through key DNA repair-associated molecules ATM, ATR, and BRCA 1/2.

PURPOSE: To study an association between IL-6 signaling and resistance to radiotherapy of prostate cancer (PCa) and explore the molecular mechanisms involved. METHODS: IL-6 expressing C4-2 and CWR22Rv1 (C4-2IL-6/CWRIL-6) and vector control (C4-2vec/CWRvec) cell lines were developed. Radiation-sensitivities of these cells were compared in clonogenic assay, Comet assay, and γH2AX staining. In xenograft animal studies, radiation-sensitivity of C4-2IL-6 cell-derived tumors vs. C4-2vec cell-derived tumors was investigated. To reveal IL-6 downstream molecules involved in DNA repair after radiation, qPCR and Western blot analyses as well as immunofluorescence staining were performed. Transcriptional control of IL-6 on ATM and ATR molecules was also investigated. RESULTS: We found C4-2IL-6 and CWRIL-6 cells survived better than their vector control cells after irradiation, and animal studies confirmed such in vitro results. We discovered that DNA repair-related molecules such as ATM, ATR, BRCA1, and BRCA2 were significantly upregulated in irradiated IL-6 expressing cells compared with vector control cells. We further defined that IL-6 signaling regulated cellular expressions of ATM and ATR at the transcriptional level through the activation of Stat3 signaling pathway. CONCLUSIONS: IL-6 leads to PCa resistance to radiation through upregulation of DNA repair associated molecules ATM, ATR, BRCA1, and BRCA2.

Radiation-induced glucocorticoid receptor promotes CD44+ prostate cancer stem cell growth through activation of SGK1-Wnt/ß-catenin signaling.

We observed cancer stem cell (CSC) population increase in radioresistant LNCaP (LNCaPR18) and C4-2 (C4-2R26) prostate cancer (PCa) cells compared with respective parental cells. Since the CD44 level increase was most significant in radioresistant PCa cells compared with parental cells among CSC markers tested, we isolated the CD44+ population from LNCaP/LNCaPR18 and C4-2/C4-2R26 cell sets via the immunomagnetic separation method and used them as CSC sources. We detected lower AR level, but higher glucocorticoid receptor (GR) level in CD44+ CSCs than CD44- non-CSCs. Higher GR level in CD44+ CSCs than CD44- cells was also detected when cells were isolated from mouse tumor tissues of LNCaPR18 cell and C4-2R26 cell-derived human xenografts and grown in culture. We then found blocking the GR signaling by adding the anti-GR agent mifepristone into the cell culture inhibited the CD44+ CSC growth while the addition of the anti-AR agent enzalutamide enhanced the CSC growth. In xenograft mouse studies in which tumors were developed from the injection of CD44+ CSCs of LNCaPR18 or C4-2R26 cell lines, retarded tumor growth in mifepristone-injected mice was observed compared with vehicle-treated mice. We next discovered the GR regulation of Wnt/ß-catenin signaling pathway. We further found that the serum/glucocorticoid regulated kinase 1 (SGK1) is the GR downstream molecule that mediates Wnt/ß-catenin signaling activation. Therefore, inhibition of either SGK1 or Wnt/ß-catenin signaling impaired the in vitro CD44+ CSC growth. From these results, we suggest that blocking GR signaling or its downstream SGK1-Wnt/ß-catenin signaling axis may suppress the radiation-induced CSC increase in PCa. KEY MESSAGES: Higher CSC population exists in radioresistant PCa cells than parental cells. Higher GR levels (and lower AR level) in CD44+ CSCs than CD44- non-CSCs. Use of anti-GR agent blocked the growth of CD44+ CSCs in in vitro/in vivo tests. GR downstream SGK1-Wnt/ß-catenin signaling axis mediates the CSC increase. Targeting this signaling axis may enhance the radiotherapy efficacy in treating PCa.

PUMA inactivation protects against oxidative stress through p21/Bcl-XL inhibition of bax death.

The tumor suppressor protein p53 activates growth arrest and proapoptotic genes in response to DNA damage. It is known that negative feedback by p21(Cip1/Waf1/Sdi1) represses p53-dependent transactivation of PUMA. The current study investigates PUMA feedback on p53 during oxidative stress from hyperoxia and the subsequent effects on cell survival mediated through p21 and Bcl-X(L). Deletion of PUMA in HCT116 colon carcinoma cells increased levels of p53 and p21, resulting in a larger G(1) population during hyperoxia. P21-dependent increase in Bcl-X(L) levels protected PUMA-deficient cells against hyperoxic cell death. Bax and Bak were both able to promote hyperoxic cell death. Bcl-X(L) protection against hyperoxic death was lost in cells lacking Bax, not PUMA, suggesting that Bcl-X(L) acts to inhibit Bax-dependent death. These results indicate that PUMA exerts a negative feedback on p53 and p21, leading to p21-dependent growth suppressive and survival changes. Enhanced survival was associated with increased Bcl-X(L) to block Bax activated cell death during oxidative stress.

Toxicity profile and pharmacokinetic study of a phase I low-dose schedule-dependent radiosensitizing paclitaxel chemoradiation regimen for inoperable non-small-cell lung cancer.

PURPOSE: We report the toxicity profile and pharmacokinetic data of a schedule-dependent chemoradiation regimen using pulsed low-dose paclitaxel for radiosensitization in a Phase I study for inoperable non-small-cell lung cancer. METHODS AND MATERIALS: Paclitaxel at escalating doses of 15 mg/m(2), 20 mg/m(2), and 25 mg/m(2) were infused on Monday, Wednesday, and Friday with daily chest radiation in cohorts of 6 patients. Daily radiation was delayed for maximal G2/M arrest and apoptotic effect, an observation from preclinical investigations. Plasma paclitaxel concentration was determined by high-performance liquid chromatography. RESULTS: Dose-limiting toxicities included 3 of 18 patients with Grade 3 pneumonitis and 3 of 18 patients with Grade 3 esophagitis. There was no Grade 4 or 5 pneumonitis or esophagitis. There was also no Grade 3 or 4 neutropenia, thrombocytopenia, anemia or neuropathy. For Dose Levels I (15 mg/m(2)), II (20 mg/m(2)), and III (25 mg/m(2)), the mean peak plasma level was 0.23 +/- 0.06 micromol/l, 0.32 +/- 0.05 micromol/l, and 0.52 +/- 0.14 micromol/l, respectively; AUC was 0.44 +/- 0.09 micromol/l, 0.61 +/- 0.1 micromol/l, and 0.96 +/- 0.23 micromol/l, respectively; and duration of drug concentration >0.05 micromol/l (t > 0.05 micromol/l) was 1.6 +/- 0.3 h, 1.9 +/- 0.2 h, and 3.0 +/- 0.9 h, respectively. CONCLUSION: Pulsed low-dose paclitaxel chemoradiation is associated with low toxicity. Pharmacokinetic data showed that plasma paclitaxel concentration >0.05 micromol/l for a minimum of 1.6 h was sufficient for effective radiosensitization.

Enhanced antitumor effect of combined triptolide and ionizing radiation.

PURPOSE: The lack of effective treatment for pancreatic cancer results in a very low survival rate. This study explores the enhancement of the therapeutic effect on human pancreatic cancer via the combination of triptolide and ionizing radiation (IR). EXPERIMENTAL DESIGN: In vitro AsPC-1 human pancreatic cancer cells were treated with triptolide alone, IR alone, or triptolide plus IR. Cell proliferation was analyzed with sulforhodamine B (SRB) method and clonogenic survival; comparison of apoptosis induced by the above treatment was analyzed by annexin V-propidium iodide (PI) staining. Furthermore, the expression of apoptotic pathway intermediates was measured by the assay of caspase activity and Western blot. Mitochondrial transmembrane potential was determined by JC-1 assay. In vivo, AsPC-1 xenografts were treated with 0.25 mg/kg triptolide, 10 Gy IR, or triptolide plus IR. The tumors were measured for volume and weight at the end of the experiment. Tumor tissues were tested for terminal nucleotidyl transferase-mediated nick end labeling (TUNEL) and immunohistochemistry. RESULTS: The combination of triptolide plus IR reduced cell survival to 21% and enhanced apoptosis, compared with single treatment. In vivo, tumor growth of AsPC-1 xenografts was reduced further in the group treated with triptolide plus IR compared with single treatment. TUNEL and immunohistochemistry of caspase-3 cleavage in tumor tissues indicated that the combination of triptolide plus IR resulted in significantly enhanced apoptosis compared with single treatments. CONCLUSIONS: Triptolide in combination with ionizing radiation produced synergistic antitumor effects on pancreatic cancer both in vitro and in vivo and seems promising in the combined modality therapy of pancreatic cancer.

Anti-cancer effect of resveratrol is associated with induction of apoptosis via a mitochondrial pathway alignment.

Resveratrol, a phytoalexin found in the skin of grapes, is believed to have multiple bioactivities including anti-cancer, anti-carcinogenesis and antiinflammatory. The mechanisms by which resveratrol might produce these effects are not well understood. In this study, malignant human pancreatic cancer cells were treated without or with resveratrol in combination with ionizing radiation (IR), and then the mitochondrial function of treated cells was evaluated using several standardized assays. They include the Calcein AM method for mitochondria transition pore; the JC-1 staining method for mitochondria membrane potential; the CM-H2DCFDA method for reactive oxygen species; and the Annexin V/propidium iodide (PI) method for apoptosis/cell death. Our results indicated that (1) pore function was partially intact after resveratrol, but resveratrol probably interfered with the accumulation of intracellular Calcein AM; (2) depolarization of the mitochondria membrane was increased in the resveratrol treated cells, consistent with mitochondrial dysfunction; (3) ROS was slightly increased with resveratrol, a phenomenon that was greatly increased when this agent was combined with IR; and (4) in parallel with the above changes in mitochondrial and drug transport, cells treated with resveratrol showed increased apoptosis as measured by Annexin V/PI staining. In summary, the anti-cancer effect of resveratrol is associated with the damage of mitochondrial function that leads to increased ROS, apoptosis, and possibly intracellular drug accumulation via inhibition of proteins involved in multi-drug resistance (MDR).