Donald R. Campbell

Crocetin inhibits pancreatic cancer cell proliferation and tumor progression in a xenograft mouse model

Crocetin, a carotenoid compound derived from saffron, has long been used as a traditional ancient medicine against different human diseases including cancer. The aim of the series of experiments was to systematically determine whether crocetin significantly affects pancreatic cancer growth both in vitro and/or in vivo. For the in vitro studies, first, MIA-PaCa-2 cells were treated with crocetin and in these sets of experiments, a proliferation assay using H3-thymidine incorporation and flow cytometric analysis suggested that crocetin inhibited proliferation. Next, cell cycle proteins were investigated. Cdc-2, Cdc-25C, Cyclin-B1, and epidermal growth factor receptor were altered significantly by crocetin. To further confirm the findings of inhibition of proliferation, H3-thymidine incorporation in BxPC-3, Capan-1, and ASPC-1 pancreatic cancer cells was also significantly inhibited by crocetin treatment. For the in vivo studies, MIA-PaCa-2 as highly aggressive cells than other pancreatic cancer cells used in this study were injected into the right hind leg of the athymic nude mice and crocetin was given orally after the development of a palpable tumor. The in vivo results showed significant regression in tumor growth with inhibition of proliferation as determined by proliferating cell nuclear antigen and epidermal growth factor receptor expression in the crocetin-treated animals compared with the controls. Both the in vitro pancreatic cancer cells and in vivo athymic nude mice tumor, apoptosis was significantly stimulated as indicated by Bax/Bcl-2 ratio. This study indicates that crocetin has a significant antitumorigenic effect in both in vitro and in vivo on pancreatic cancer. [Mol Cancer Ther 2009;8(2):315–23]

Biphasic estrogen response on bovine adrenal medulla capillary endothelial cell adhesion, proliferation and tube formation

Abnormal angiogenesis underlies many pathological conditions and is critical for the growth and maintenance of various types of tumors, including hormone-dependent cancers. Since estrogens are potent carcinogens in humans and rodents, and are involved in regulating angiogenesis, this study was designed to examine the effect of estrogen on the behavior of an established bovine capillary endothelial cell line, a simple and physiologically relevant model of the capillary wall. The results demonstrate that 17β-estradiol (E2), at different conditions, exerts both stimulatory and inhibitory effects on endothelial cell adhesion, proliferation and tube formation in vitro. Utilizing a cellular attachment assay, chronic exposure to nanomolar concentrations of E2 (i.e. 1 and 10 nM) increased endothelial cell adhesion significantly compared to vehicle treated controls. Cellular adhesion was inhibited by micromolar concentrations of E2. Cell count, PCNA immunohistochemistry and Western blot analysis demonstrated enhanced cell proliferation at low E2 concentration in estrogen-deplete medium. Inhibition of cellular proliferation was observed in both estrogen-replete and deplete medium at higher E2 concentrations (i.e. 1 and 10 µM). Furthermore, in vitro tube formation increased up to 3.0 fold in the presence of 10 nM and higher E2 concentrations. The present observations indicate that in vitro regulation of capillary endothelial cell adhesion, proliferation and capillary tube formation by estrogen, are dose dependent.

Cysteine-rich 61-connective tissue growth factor-nephroblastoma-overexpressed 5 (CCN5)/Wnt-1-induced signaling protein-2 (WISP-2) regulates microRNA-10b via hypoxia-inducible factor-1α-TWIST signaling networks in human breast cancer cells.

Background: Because CCN5 is an anti-invasive gene, present studies were designed to determine whether CCN5 exerts its anti-invasive function through controlling microRNA-10b expression. Results: Up-regulation of TWIST1, a miR-10b activator, can be achieved by CCN5 silencing through the activation of HIF-1α JNK signaling. Conclusion: CCN5 is a negative regulator of miR-10b in breast cancer cells. Significance: The reactivation of CCN5 could be a unique therapeutic strategy for Triple negative breast cancer. MicroRNAs (miRNAs) are naturally occurring single-stranded RNA molecules that post-transcriptionally regulate the expression of target mRNA transcripts. Many of these target mRNA transcripts are involved in regulating processes commonly altered during tumorigenesis and metastatic growth. These include cell proliferation, differentiation, apoptosis, migration, and invasion. Among the several miRNAs, miRNA-10b (miR-10b) expression is increased in metastatic breast cancer cells and positively regulates cell migration and invasion through the suppression of the homeobox D10 (HOXD10) tumor suppressor signaling pathway. In breast metastatic cells, miR-10b expression is enhanced by a transcription factor TWIST1. We find that miR-10b expression in breast cancer cells can be suppressed by CCN5, and this CCN5 effect is mediated through the inhibition of TWIST1 expression. Moreover, CCN5-induced inhibition of TWIST1 expression is mediated through the translational inhibition/modification of hypoxia-inducible factor-1α via impeding JNK signaling pathway. Collectively, these studies suggest a novel regulatory pathway exists through which CCN5 exerts its anti-invasive function. On the basis of these findings, it is plausible that reactivation of CCN5 in miR-10b-positive invasive/metastatic breast cancers alone or in combination with current therapeutic regimens could provide a unique, alternative strategy to existing breast cancer therapy.

Effect of H. pylori status on gastric ulcer healing in patients continuing nonsteroidal anti-inflammatory therapy and receiving treatment with lansoprazole or ranitidine

OBJECTIVES:The purpose of this research was to determine the impact of pretreatment Helicobacter pylori infection on gastric ulcer healing rates in patients receiving nonsteroidal anti-inflammatory drugs (NSAIDs) and antisecretory medications.METHODS:This was a pooled, prospective analysis of two identical double blind, multicenter, parallel group studies. Six hundred ninety-two patients receiving NSAIDs and with endoscopy-documented gastric ulcers were enrolled from 90 North American sites in primary care and referral centers. Patients were randomized to receive ranitidine (150 mg b.i.d.) or lansoprazole (15 mg or 30 mg once daily) for 8 wk. Ulcer healing was assessed by endoscopy at 4 and 8 wk in an intent-to-treat population. H. pylori status was determined at baseline by histology.RESULTS:Across all three treatment groups, gastric ulcers were more likely to heal and heal faster if the individual was infected with H. pylori. Healing rates at 8 wk were statistically significantly greater among H. pylori positive patients (n = 181) than among negative patients (n = 497) (70% vs 61%, respectively; p < 0.05), especially among those with large ulcers (>10 mm) and in younger patients (<60 yr old). Simple healing rates (regardless of H. pylori status) were significantly better in the 15- and 30-mg lansoprazole groups than in the ranitidine group after 4 wk (46%, 54%, and 32%, respectively; p≤ 0.01) and 8 wk (66%, 74%, and 50%, respectively; p < 0.001).CONCLUSIONS:In patients receiving NSAIDs, gastric ulcer healing with an antisecretory agent is significantly enhanced in the presence of H. pylori infection.

Tumor angiogenesis in chronic pancreatitis and pancreatic adenocarcinoma: impact of K-ras mutations.

Chronic pancreatitis (CP) is one condition in which epidemiologic studies have demonstrated a definite association with pancreatic adenocarcinoma (PAC). The pathophysiologic and molecular events that either predispose to the development of, or potentiate the growth of, PAC are unknown. Mutation of the codon 12 K-ras gene is one genetic aberration commonly associated with development of PAC. Tumor angiogenesis, or microvascular proliferation of new capillaries, is another pathophysiologic alteration associated with PAC. Although activated ras oncogenes modulate tumor angiogenesis/neovascularization in some tumors, the importance of tumor angiogenesis and the role of K-ras mutation in regulating angiogenesis in CP and PAC are unknown. The aim of this study was to elucidate the relationship between angiogenesis and K-ras mutations in CP and PAC. Tumor angiogenesis and K-ras mutations were evaluated in resected specimens from 25 CP (23 CP plus two CP with PAC) and 16 PAC patients. Tumor angiogenesis was determined using immunohistochemistry of factor VIII–related antigen (FVIIIRAg) and ras mutations were identified by enriched-nested polymerase chain reaction. The mean number of FVIIIRAg-positive blood vessels was significantly (p < 0.005) higher in PAC (23.0 ± 7.5), CP with a mutant K-ras genome (17.7 ± 2.8) and CP with a normal K-ras genome (6.5 ± 3.8), compared to unaffected areas. Codon 12 K-ras mutations were detected in three of 25 CP specimens (12%) and in 15 of 16 PAC specimens (94%). In CP patients with mutant K-ras in their genome, microvessel density was significantly (p < 0.01) elevated, compared to patients with a normal K-ras genome. Statistical analyses (Spearman rank-difference correlation coefficient, Student t test, and &khgr;2 analysis) indicated a significant association between codon 12 K-ras mutations and tumor angiogenesis in both CP and PAC. This study demonstrates a significant association between angiogenesis and K-ras mutation in both PAC and CP. At a minimum, K-ras mutation is associated with the events that increase angiogenesis and it may potentiate or promote tumor angiogenesis.

A Two-Step Enriched-Nested PCR Technique Enhances Sensitivity for Detection of Codon 12 K-ras Mutations in Pancreatic Adenocarcinoma

Mutations at codon 12 of the K-ras gene have been detected in pancreatic adenocarcinomas by a variety of techniques. A few of these techniques are very sensitive, identifying the mutations in 96–100% of cases. However, these sensitive techniques are labor intensive, utilizing multistep processing and radioactive material. Much simpler techniques, involving nonradioactive single-step polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) have been employed to detect K-ras mutations at codon 12 in pancreatic adenocarcinomas. However, the low sensitivity of these single-step PCR/RFLP techniques is unacceptable. A simple and nonradioactive PCR/RFLP-based method for detection of K-ras codon 12 mutations in formalin-fixed, paraffin-embedded tissue sections of pancreatic adenocarcinoma is described and compared to the traditional PCR technique. K-ras gene mutations at codon 12 were detected by a modified two-step enrich-nested PCR (EN-PCR)/RFLP method, and their existence was confirmed by direct DNA sequencing analysis of the product. When the two-step EN-PCR/RFLP technique was compared to the single-step PCR/RFLP method, K-ras codon 12 mutations were detected in 100% of pancreatic adenocarcinomas (15/15) with the EN-PCR/RFLP method, while half as many (9/15) were detected with the single-step PCR/RFLP method. This study demonstrates that the sensitivity of the simple two-step EN-PCR/RFLP technique is comparable to that of the more complex methods for detecting K-ras mutations at codon 12 in formalin-fixed, paraffin-embedded tissue sections of pancreatic adenocarcinoma and its sensitivity is superior to that of the single-step technique.

Specificity of polymerase chain reaction monoclonality for diagnosis of gastric mucosa-associated lymphoid tissue (MALT) lymphoma: direct comparison to Southern blot gene rearrangement.

The specificity of polymerase chain reactionmonoclonality in the diagnosis of gastric lymphoma wasprospectively evaluated. Gastric mucosal tissue fromnormal gastric mucosa (N = 13), benign gastric ulcers (N = 3), chronic Helicobacter pylori gastritis(N = 3), gastric mucosa-associated lymphoid tissue (N =16), and gastric lymphoma (N = 15) was obtained.Polymerase chain reaction amplification of theheavy-chain framework 2A gene was performed. Thesensitivity and specificity of heavy-chain clonality, inthe detection of gastric lymphoma, were 73.3% and 45.7%,respectively. Determination of monoclonality bypolymerase chain reaction methodology is not an acceptabletechnique for confirming the diagnosis of gastriclymphoma as it is too sensitive, detecting minutepopulations of clonal lymphocytes that occur in benign diseases as well as larger populations ofclonal lymphocytes associated with malignant gastriclymphoproliferative diseases. Southern blot generearrangement testing should be utilized to determineclonality in the evaluation of gastric lymphocyticinfiltrates.

Double-Blind Comparison of Lansoprazole 15 mg, Lansoprazole 30 mg, and Placebo in the Maintenance of Healed Gastric Ulcer

Our purpose was to compare the safety andefficacy of lansoprazole 15 mg and 30 mg with placebo inpreventing recurrence in 49 patients with a history ofgastric ulcer. Within one month, 40% of patients receiving placebo experienced ulcer recurrencecompared to 0% and 7% of patients receiving lansoprazole15 mg and 30 mg, respectively. All placebo patientsbecame symptomatic, experienced ulcer recurrence or withdrew from the study by month 9. Ascompared to placebo, a significantly (P < 0.001)higher percentage of patients treated with lansoprazole15 mg (83%) and lansoprazole 30 mg (93%) with healed gastric ulcer disease remained healed at month12. Of patients asymptomatic at baseline, 100% and 59%of those treated with lansoprazole 15 mg and 30 mg,respectively, remained asymptomatic at month 12. The incidence of adverse events was comparableamong the treatment groups. Lansoprazole safely andeffectively reduces ulcer recurrence in patients with ahistory of gastric ulcer disease.

Human pancreatic cancer progression: an anarchy among CCN-siblings

Decades of basic and translational studies have identified the mechanisms by which pancreatic cancer cells use molecular pathways to hijack the normal homeostasis of the pancreas, promoting pancreatic cancer initiation, progression, and metastasis, as well as drug resistance. These molecular pathways were explored to develop targeted therapies to prevent or cure this fatal disease. Regrettably, the studies found that majority of the molecular events that dictate carcinogenic growth in the pancreas are non-actionable (potential non-responder groups of targeted therapy). In this review we discuss exciting discoveries on CCN-siblings that reveal how CCN-family members contribute to the different aspects of the development of pancreatic cancer with special emphasis on therapy.

Epidermal growth factor receptor: is a novel therapeutic target for pancreatic cancer?

Expression of epidermal growth factor receptors (EGFR) is exaggerated in pancreatic adenocarcinoma and activation of EGFR appears to have an important role in the growth and differentiation of this and in other tumors. Therefore, blockade or inactivation of EGFR by monoclonal antibodies or by tyrosine kinase inhibitors has significant potential as an effective anti-cancer therapy. One of the very recent significant developments in the field of molecular biology involves the use of antisense of EGFR or EGFR gene silencing in pancreatic cancer cells as a potential targeted therapy for patients with pancreatic adenocarcinoma.