Huangbing Wu

Vascular endothelial growth factor, a novel and highly accurate pancreatic fluid biomarker for serous pancreatic cysts

BACKGROUND Mucinous pancreatic cysts (intraductal papillary mucinous neoplasm and mucinous cystic neoplasm) have the potential to progress to invasive pancreatic adenocarcinoma, presenting an opportunity for early detection, prevention, and cure. Serous cystic neoplasms (SCN) have no malignant potential, but can mimic mucinous pancreatic cysts on imaging. Therefore, identification of biomarkers that can distinguish between cystic lesions is critically important. We hypothesize that vascular endothelial growth factor (VEGF)-A levels in pancreatic fluid correlate with pathologic diagnosis. STUDY DESIGN Pancreatic cyst/duct fluid samples were prospectively collected from patients undergoing pancreatic resection and correlated with surgical pathology. VEGF levels were detected by ELISA. VEGF-A and VEGF receptor 2 expression in pancreatic tissue was localized by immunohistochemistry. Genetic alterations of the von Hippel-Lindau gene were determined by targeted next-generation sequencing. RESULTS Eighty-seven patients met inclusion criteria for enrollment. Final pathologic diagnoses included pseudocyst (n = 9), SCN (n = 17), mucinous cystic neoplasm (n = 24), low/moderate grade intraductal papillary mucinous neoplasm (n = 16), high-grade/invasive intraductal papillary mucinous neoplasm (n = 10), and pancreatic ductal adenocarcinoma (n = 11). VEGF-A was significantly upregulated in SCN cyst fluid compared with all other diagnoses (p < 0.0001). With a cut-off of 8,500 pg/mL, VEGF-A has 100% sensitivity and 97% specificity as an SCN biomarker. VEGF-A and VEGF receptor 2 are overexpressed in SCN cyst tissue. VEGF-C was also significantly elevated in SCN cyst fluid (p < 0.0001). With a cut-off set at 200 pg/mL, VEGF-C identifies SCN with 100% sensitivity and 90% specificity. The presence of a von Hippel-Lindau mutation in SCN cyst tissue correlates with elevated cyst fluid VEGF levels. CONCLUSIONS This is the first report of a cyst fluid protein biomarker that can positively identify SCN. The ability to distinguish SCN from premalignant/malignant pancreatic cysts can spare the cost and risk of surveillance and surgical intervention in select patients.

Suppression of pancreatic tumor growth by combination chemotherapy with sulindac and LC-1 is associated with cyclin D1 inhibition in vivo.

The design of novel targeted or combination therapies may improve treatment options for pancreatic cancer. Two targets of recent interest are nuclear factor-κB (NF-κB) and cyclooxygenase (COX), known to be activated or overexpressed, respectively, in pancreatic cancer. We have previously shown that parthenolide, a proapoptotic drug associated with NF-κB inhibition, enhanced the growth suppression of pancreatic cancer cells by the COX inhibitor sulindac in vitro. In the present study, a bioavailable analogue of parthenolide, LC-1, and sulindac were evaluated in vivo using a xenograft model of human pancreatic cancer. Treatment groups included placebo, low-dose/high-dose LC-1 (20 and 40 mg/kg), low-dose/high-dose sulindac (20 and 60 mg/kg), and low-dose combination LC-1/sulindac (20 mg/kg each). In MiaPaCa-2 xenografts, tumor growth was inhibited by either high-dose sulindac or LC-1. In BxPC-3 xenografts, tumor size was significantly reduced by treatment with the low-dose LC-1/sulindac combination or high-dose sulindac alone (P < 0.05). Immunohistochemistry of BxPC-3 tumors revealed a significant decrease in Ki-67 and CD31 staining by high-dose sulindac, with no significant changes in COX-1/COX-2 levels or activity in any of the treatment groups. NF-κB DNA-binding activity was significantly decreased by high-dose LC-1. Cyclin D1 protein levels were reduced by the low-dose LC-1/sulindac combination or high-dose sulindac alone, correlating with BxPC-3 tumor suppression. These results suggest that LC-1 and sulindac may mediate their antitumor effects, in part, by altering cyclin D1 levels. Furthermore, this study provides preclinical evidence for the therapeutic efficacy of these agents. [Mol Cancer Ther 2007;6(6):1736–44]

Effect of celecoxib and the novel anti-cancer agent, dimethylamino-parthenolide, in a developmental model of pancreatic cancer.

Objectives: Cancer of the exocrine pancreas is the fourth leading cause of cancer-related deaths in the United States. The efficacy of a novel bioavailable anticancer agent, dimethylamino-parthenolide (DMAPT), and the cyclooxygenase 2 inhibitor, celecoxib, was evaluated in a carcinogen-induced developmental model of pancreatic cancer. Methods: Syrian golden hamsters were injected with N-nitrosobis(2-oxopropyl)amine, once weekly for 6 weeks. Upon the first injection, hamsters were randomized as follows: placebo, low-/high-dose DMAPT (20 and 40 mg/kg per day), low-/high-dose celecoxib (10and 50 mg/kg per day), or combination DMAPT/celecoxib (low/low, high/high). Results: The 32-week trial showed that 40 mg/kg DMAPT alone significantly decreased the size of gross pancreatic cancers relative to placebo. No significant difference in gross tumor number was observed between the treatment groups and placebo with the exception of 50 mg/kg celecoxib with a higher tumor incidence; this group also exhibited lower lymphotactin levels suggestive of decreased immune surveillance. Tumor invasion into adjacent organs and metastasis were not observed in the DMAPT/celecoxib treatment groups. Drug targets including prostaglandin E2, prostaglandin E2 metabolite and activated nuclear factor &kgr;B were significantly decreased. Conclusions: Dimethylamino-parthenolide and celecoxib have the potential to be novel chemotherapeutic agents for pancreatic cancer; however, further optimization or the use of other modalities may be required for chemoprevention.

Targeting mitogen-activated protein kinase kinase with the inhibitor PD0325901 decreases hepatocellular carcinoma growth in vitro and in mouse model systems†

Hepatocellular carcinoma (HCC) is a common cause of death from solid organ malignancy worldwide. Extracellular signal‐regulated/mitogen‐activated protein kinase kinase (MEK) signaling is a critical growth regulatory pathway in HCC. Targeting MEK with a novel small molecule inhibitor, PD0325901, may inhibit HCC tumorigenesis. PD0325901 (0.01‐100 nM) inhibited growth and MEK activity in vitro in immortalized murine transforming growth factor alpha (TGF‐α) transgenic hepatocyte (TAMH) cells, derived from the livers of TGF‐α transgenic mice. Treatment of athymic mice bearing TAMH flank tumors with vehicle or PD0325901 (20 mg/kg) revealed a significant reduction of MEK activity ex vivo 24 hours after a single PD0325901 dose. The growth rate of TAMH flank tumors over 16 days was reduced threefold in the treatment arm (1113 ± 269% versus 3077 ± 483%, P < 0.01). PD0325901 exhibited similar inhibitory effects in HepG2 and Hep3B human HCC cells in vitro and in Hep3B flank tumors in vivo. To confirm this in a developmental model, MT‐42 (CD‐1) TGF‐α mice were treated with vehicle or PD0325901 (20 mg/kg) for 5 weeks. Gross HCC was detected in 47% and 13.3% of the control and treatment mice, respectively. Tumor growth suppression by PD0325901 relative to vehicle was also shown by magnetic resonance imaging. These studies provide compelling preclinical evidence that targeting MEK in human clinical trials may be promising for the treatment of HCC. (HEPATOLOGY 2010.)

Tailored α-methylene-γ-butyrolactones and their effects on growth suppression in pancreatic carcinoma cells.

A selected series of racemic α-methylene-γ-butyrolactones (AMGBL) were synthesized via allylboration and screened against three human pancreatic cancer cell lines (Panc-1, MIA PaCa-2, and BxPC-3). This systematic study established a discernible relationship between the substitution pattern of AMGBL and their anti-proliferative activity. β,γ-diaryl-AMGBLs, particularly those with a trans-relationship exhibited higher potency than parthenolide and LC-1 against all three cell lines.

Dimethylaminoparthenolide and gemcitabine: a survival study using a genetically engineered mouse model of pancreatic cancer

BackgroundPancreatic cancer remains one of the deadliest cancers due to lack of early detection and absence of effective treatments. Gemcitabine, the current standard-of-care chemotherapy for pancreatic cancer, has limited clinical benefit. Treatment of pancreatic cancer cells with gemcitabine has been shown to induce the activity of the transcription factor nuclear factor-kappaB (NF-κB) which regulates the expression of genes involved in the inflammatory response and tumorigenesis. It has therefore been proposed that gemcitabine-induced NF-κB activation may result in chemoresistance. We hypothesize that NF-κB suppression by the novel inhibitor dimethylaminoparthenolide (DMAPT) may enhance the effect of gemcitabine in pancreatic cancer.MethodsThe efficacy of DMAPT and gemcitabine was evaluated in a chemoprevention trial using the mutant Kras and p53-expressing LSL-KrasG12D/+; LSL-Trp53R172H; Pdx-1-Cre mouse model of pancreatic cancer. Mice were randomized to treatment groups (placebo, DMAPT [40 mg/kg/day], gemcitabine [50 mg/kg twice weekly], and the combination DMAPT/gemcitabine). Treatment was continued until mice showed signs of ill health at which time they were sacrificed. Plasma cytokine levels were determined using a Bio-Plex immunoassay. Statistical tests used included log-rank test, ANOVA with Dunnett’s post-test, Student’s t-test, and Fisher exact test.ResultsGemcitabine or the combination DMAPT/gemcitabine significantly increased median survival and decreased the incidence and multiplicity of pancreatic adenocarcinomas. The DMAPT/gemcitabine combination also significantly decreased tumor size and the incidence of metastasis to the liver. No significant differences in the percentages of normal pancreatic ducts or premalignant pancreatic lesions were observed between the treatment groups. Pancreata in which no tumors formed were analyzed to determine the extent of pre-neoplasia; mostly normal ducts or low grade pancreatic lesions were observed, suggesting prevention of higher grade lesions in these animals. While gemcitabine treatment increased the levels of the inflammatory cytokines interleukin 1α (IL-1α), IL-1β, and IL-17 in mouse plasma, DMAPT and DMAPT/gemcitabine reduced the levels of the inflammatory cytokines IL-12p40, monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-1 beta (MIP-1β), eotaxin, and tumor necrosis factor-alpha (TNF-α), all of which are NF-κB target genes.ConclusionIn summary, these findings provide preclinical evidence supporting further evaluation of agents such as DMAPT and gemcitabine for the prevention and treatment of pancreatic cancer.

Efficacy of dimethylaminoparthenolide and sulindac in combination with gemcitabine in a genetically engineered mouse model of pancreatic cancer

Objectives Pancreatic cancer remains one of the deadliest diseases, with limited surgical and treatment options. Two targets of interest include the transcription factor nuclear factor-&kgr;B and cyclooxygenase-2, which are constitutively activated and overexpressed, respectively, in human pancreatic adenocarcinoma. We have previously shown that dimethylaminoparthenolide (DMAPT), a bioavailable nuclear factor-&kgr;B inhibitor, and the cyclooxygenase inhibitors sulindac and celecoxib have potential chemotherapeutic efficacy. The current study evaluates the efficacy of intervention with DMAPT and sulindac in the LSL-KrasG12D;Pdx-1-Cre genetically engineered mouse model. Gemcitabine, traditionally a chemotherapeutic agent, has relatively low toxicity; thus, combinations with low-dose gemcitabine were also explored. Methods LSL-KrasG12D;Pdx-1-Cre mice at 7 months of age were randomized into placebo, DMAPT (40 mg/kg per day), sulindac (20 mg/kg per day), gemcitabine (50 mg/kg twice weekly), and combination treatment groups. After 3 months of treatment, the mice were killed. Results The percentage of normal pancreatic ducts was significantly increased by the combinations of DMAPT/sulindac, DMAPT/gemcitabine, sulindac/gemcitabine, and DMAPT/sulindac/gemcitabine compared to placebo. Additionally, the percentage of mouse pancreatic intraepithelial neoplasia-2 lesions was significantly decreased by DMAPT/gemcitabine. Conclusions Intervention with DMAPT and sulindac in combination with gemcitabine may delay or prevent progression of premalignant pancreatic lesions in the LSL-KrasG12D;Pdx-1-Cre mouse model of pancreatic cancer.

Ethanol-TGFα-MEK Signaling Promotes Growth of Human Hepatocellular Carcinoma

BACKGROUND Chronic ethanol intake is a significant risk factor for the development of cirrhosis and hepatocellular carcinoma (HCC). The effects of ethanol on extracellular signal-regulated kinase (ERK) activation, transforming growth factor alpha (TGF-alpha), and HCC growth were examined in this study. METHODS HepG2, SKHep, Hep3B human HCC cells, or normal human hepatocytes were treated with ethanol (0-100 mM), exogenous TGF-alpha, TGF-alpha neutralization antibody or the MEK inhibitor U0126. TGF-alpha levels were quantified by ELISA. Growth was determined by trypan blue-excluded cell counts. Cell cycle phase distribution was determined by flow cytometry. Protein expression was determined by Western blot. RESULTS Ethanol treatment (10-40 mM) increased ERK activation in HepG2 and SKHep HCC cells but not in Hep3B or human hepatocyte cells. Growth increased in HepG2 (174 +/- 29%, P < 0.05) and SKHep (149 +/- 12%, P < 0.05) cells in response to ethanol treatment. Correspondingly, ethanol increased S phase distribution in these cells. U0126 suppressed ethanol-induced growth increases. Ethanol treatment for 24 h also raised TGF-alpha levels in HepG2 cells (118%-198%) and SKHep cells (112%-177%). Exogenous administration of recombinant TGF-alpha mimicked the ethanol-induced growth in HepG2 and SKHep cells; TGF-alpha neutralization antibody effectively abrogated this effect. The TGF-a neutralization antibody also prevented ERK activation by ethanol in HepG2 cells. CONCLUSIONS These data demonstrate that clinically relevant doses of ethanol stimulate ERK-dependent proliferation of HCC cells. Ethanol up-regulates TGF-alpha levels in HCC cells and enhances growth through cell cycles changes, which appear to be mediated through TGF-alpha-MEK-ERK signaling. Ethanol-MEK signaling in normal hepatocytes is absent, suggesting that ethanol promotion of HCC growth may in part depend upon the acquisition of cancer-specific signaling by hepatocytes.

Pancreatic cyst fluid glucose: rapid, inexpensive, and accurate diagnosis of mucinous pancreatic cysts

Background. The most widely accepted biochemical test for preoperative differentiation of mucinous from benign, nonmucinous pancreatic cysts is cyst fluid carcinoembryonic antigen. However, the diagnostic accuracy of carcinoembryonic antigen ranges from 70% to 86%. Based on previous work, we hypothesize that pancreatic cyst fluid glucose may be an attractive alternative to carcinoembryonic antigen. Methods. Pancreatic cyst fluid was collected during endoscopic or operative intervention. Diagnoses were pathologically confirmed. Glucose and carcinoembryonic antigen were measured using a patient glucometer and automated analyzer/enzyme‐linked immunosorbent assay. Sensitivity, specificity, accuracy, and receiver operator characteristic analyses were performed. Results. Cyst fluid samples from 153 patients were evaluated (mucinous: 25 mucinous cystic neoplasms, 77 intraductal papillary mucinous neoplasms, 4 ductal adenocarcinomas; nonmucinous: 21 serous cystic neoplasms, 9 cystic neuroendocrine tumors, 14 pseudocysts, 3 solid pseudopapillary neoplasms). Median cyst fluid glucose was lower in mucinous versus nonmucinous cysts (19 vs 96 mg/dL; P < .0001). With a threshold of ≤ 50 mg/dL, cyst fluid glucose was 92% sensitive, 87% specific, and 90% accurate in diagnosing mucinous pancreatic cysts. In comparison, cyst fluid carcinoembryonic antigen with a threshold of >192 ng/mL was 58% sensitive, 96% specific, and 69% accurate. Area under the curve for glucose and CEA were similar at 0.91 and 0.92. Conclusion. Cyst fluid glucose has significant advantages over carcinoembryonic antigen and should be considered for use as a routine diagnostic test for pancreatic mucinous cysts.

Circulating Thrombospondin-2 enhances prediction of malignant intraductal papillary mucinous neoplasm

BACKGROUND IPMNs are cystic pancreatic lesions with variable malignant potential. Thrombospondin-2 (THBS2)-an endogenous, anti-angiogenic matrix glycoprotein-may modulate tumor progression. We hypothesized that circulating levels of THBS2 could aid in preoperative prediction of malignant IPMN. METHODS Preoperative serum/plasma samples were procured from patients undergoing surgery. Circulating levels of THBS2 were measured (enzyme-linked immunosorbent assay) and compared to surgical pathology IPMN dysplastic grade. RESULTS 164 patients underwent THBS2 testing (100 Low/Moderate-IPMN; 64 High-Grade/Invasive-IPMN). Circulating THBS2 (mean ± SD) was greater in High-Grade/Invasive-IPMN than Low/Moderate-grade IPMN (26.6 ± 12.7 ng/mL vs. 20.4 ± 8.2 ng/mL; P < 0.001). THBS2 (AUC = 0.65) out-performed CA19-9 (n = 144; AUC = 0.59) in predicting IPMN grade. The combination of THBS2, CA19-9, radiographic main-duct involvement, main-duct diameter, age, sex, and BMI (AUC 0.82; n = 137) provided a good prediction model for IPMN grade. CONCLUSION Circulating THBS2 is correlated with IPMN dysplasia grade. THBS2 alone did not strongly predict IPMN grade but rather strengthened prediction models for High-Grade/Invasive IPMN when combined with other clinical/biomarker data.