Biological effects of a de novo designed myxoma virus peptide analogue: evaluation of cytotoxicity on tumor cells

Background

The Resonant Recognition Model (RRM) is a physico-mathematical model that interprets protein sequence linear information using digital signal processing methods. In this study the RRM concept was employed for structure-function analysis of myxoma virus (MV) proteins and the design of a short bioactive therapeutic peptide with MV-like antitumor/cytotoxic activity.

Methodology/Principal Findings

The analogue RRM-MV was designed by RRM as a linear 18 aa 2.3 kDa peptide. The biological activity of this computationally designed peptide analogue against cancer and normal cell lines was investigated. The cellular cytotoxicity effects were confirmed by confocal immunofluorescence microscopy, by measuring the levels of cytoplasmic lactate dehydrogenase (LDH) and by Prestoblue cell viability assay for up to 72 hours in peptide treated and non-treated cell cultures. Our results revealed that RRM-MV induced a significant dose and time-dependent cytotoxic effect on murine and human cancer cell lines. Yet, when normal murine cell lines were similarly treated with RRM-MV, no cytotoxic effects were observed. Furthermore, the non-bioactive RRM designed peptide RRM-C produced negligible cytotoxic effects on these cancer and normal cell lines when used at similar concentrations. The presence/absence of phosphorylated Akt activity in B16F0 mouse melanoma cells was assessed to indicate the possible apoptosis signalling pathway that could be affected by the peptide treatment. So far, Akt activity did not seem to be significantly affected by RRM-MV as is the case for the original viral protein.

Conclusions/Significance

Our findings indicate the successful application of the RRM concept to design a bioactive peptide analogue (RRM-MV) with cytotoxic effects on tumor cells only. This 2.345 kDa peptide analogue to a 49 kDa viral protein may be suitable to be developed as a potential cancer therapeutic. These results also open a new direction to the rational design of therapeutic agents for future cancer treatment.     

Tenascin-C enhances pancreatic cancer cell growth and motility and affects cell adhesion through activation of the integrin pathway

Background

Pancreatic cancer (PDAC) is characterized by an abundant fibrous tissue rich in Tenascin-C (TNC), a large ECM glycoprotein mainly synthesized by pancreatic stellate cells (PSCs). In human pancreatic tissues, TNC expression increases in the progression from low-grade precursor lesions to invasive cancer. Aim of this study was the functional characterization of the effects of TNC on biologic relevant properties of pancreatic cancer cells.

Methods

Proliferation, migration and adhesion assays were performed on pancreatic cancer cell lines treated with TNC or grown on a TNC-rich matrix. Stable transfectants expressing the large TNC splice variant were generated to test the effects of endogenous TNC. TNC-dependent integrin signaling was investigated by immunoblotting, immunofluorescence and pharmacological inhibition.

Results

Endogenous TNC promoted pancreatic cancer cell growth and migration. A TNC-rich matrix also enhanced migration as well as the adhesion to the uncoated growth surface of poorly differentiated cell lines. In contrast, adhesion to fibronectin was significantly decreased in the presence of TNC. The effects of TNC on cell adhesion were paralleled by changes in the activation state of paxillin and Akt.

Conclusion

TNC affects proliferation, migration and adhesion of poorly differentiated pancreatic cancer cell lines and might therefore play a role in PDAC spreading and metastasis in vivo.     

Specific Targeting of Caspase-9/PP2A Interaction as Potential New Anti-Cancer Therapy

Purpose

PP2A is a serine/threonine phosphatase critical to physiological processes, including apoptosis. Cell penetrating peptides are molecules that can translocate into cells without causing membrane damage. Our goal was to develop cell-penetrating fusion peptides specifically designed to disrupt the caspase-9/PP2A interaction and evaluate their therapeutic potential in vitro and in vivo.

Experimental Design

We generated a peptide containing a penetrating sequence associated to the interaction motif between human caspase-9 and PP2A (DPT-C9h), in order to target their association. Using tumour cell lines, primary human cells and primary human breast cancer (BC) xenografts, we investigated the capacity of DPT-C9h to provoke apoptosis in vitro and inhibition of tumour growth (TGI) in vivo. DPT-C9h was intraperitonealy administered at doses from 1 to 25 mg/kg/day for 5 weeks. Relative Tumour Volume (RTV) was calculated.

Results

We demonstrated that DPT-C9h specifically target caspase-9/PP2A interaction in vitro and in vivo and induced caspase-9-dependent apoptosis in cancer cell lines. DPT-C9h also induced significant TGI in BC xenografts models. The mouse-specific peptide DPT-C9 also induced TGI in lung (K-Ras model) and breast cancer (PyMT) models. DPT-C9h has a specific effect on transformed B cells isolated from chronic lymphocytic leukemia patients without any effect on primary healthy cells. Finally, neither toxicity nor immunogenic responses were observed.

Conclusion

Using the cell-penetrating peptides blocking caspase-9/PP2A interactions, we have demonstrated that DPT-C9h had a strong therapeutic effect in vitro and in vivo in mouse models of tumour progression.     

c-Myc Is Required for Maintenance of Glioma Cancer Stem Cells

Background

Malignant gliomas rank among the most lethal cancers. Gliomas display a striking cellular heterogeneity with a hierarchy of differentiation states. Recent studies support the existence of cancer stem cells in gliomas that are functionally defined by their capacity for extensive self-renewal and formation of secondary tumors that phenocopy the original tumors. As the c-Myc oncoprotein has recognized roles in normal stem cell biology, we hypothesized that c-Myc may contribute to cancer stem cell biology as these cells share characteristics with normal stem cells.

Methodology/Principal Findings

Based on previous methods that we and others have employed, tumor cell populations were enriched or depleted for cancer stem cells using the stem cell marker CD133 (Prominin-1). We characterized c-Myc expression in matched tumor cell populations using real time PCR, immunoblotting, immunofluorescence and flow cytometry. Here we report that c-Myc is highly expressed in glioma cancer stem cells relative to non-stem glioma cells. To interrogate the significance of c-Myc expression in glioma cancer stem cells, we targeted its expression using lentivirally transduced short hairpin RNA (shRNA). Knockdown of c-Myc in glioma cancer stem cells reduced proliferation with concomitant cell cycle arrest in the G₀/G₁ phase and increased apoptosis. Non-stem glioma cells displayed limited dependence on c-Myc expression for survival and proliferation. Further, glioma cancer stem cells with decreased c-Myc levels failed to form neurospheres in vitro or tumors when xenotransplanted into the brains of immunocompromised mice.

Conclusions/Significance

These findings support a central role of c-Myc in regulating proliferation and survival of glioma cancer stem cells. Targeting core stem cell pathways may offer improved therapeutic approaches for advanced cancers.     

Purine Nucleoside Phosphorylase Targeted by Annexin V to Breast Cancer Vasculature for Enzyme Prodrug Therapy

Background and Purpose

The targeting of therapeutics is a promising approach for the development of new cancer treatments that seek to reduce the devastating side effects caused by the systemic administration of current drugs. This study evaluates a fusion protein developed as an enzyme prodrug therapy targeted to the tumor vasculature. Cytotoxicity would be localized to the site of the tumor using a protein fusion of purine nucleoside phosphorylase (PNP) and annexin V. Annexin V acts as the tumor-targeting component of the fusion protein as it has been shown to bind to phosphatidylserine expressed externally on cancer cells and the endothelial cells of the tumor vasculature, but not normal vascular endothelial cells. The enzymatic component of the fusion, PNP, converts the FDA-approved cancer therapeutic, fludarabine, into a more cytotoxic form. The purpose of this study is to determine if this system has a good potential as a targeted therapy for breast cancer.

Methods

A fusion of E. coli purine nucleoside phosphorylase and human annexin V was produced in E. coli and purified. Using human breast cancer cell lines MCF-7 and MDA-MB-231 and non-confluent human endothelial cells grown in vitro, the binding strength of the fusion protein and the cytotoxicity of the enzyme prodrug system were determined. Endothelial cells that are not confluent expose phosphatidylserine and therefore mimic the tumor vasculature.

Results

The purified recombinant fusion protein had good enzymatic activity and strong binding to the three cell lines. There was significant cell killing (p<0.001) by the enzyme prodrug treatment for all three cell lines, with greater than 80% cytotoxicity obtained after 6 days of treatment.

Conclusion

These results suggest that this treatment could be useful as a targeted therapy for breast cancer.     

Immunolocalization of the Tumor-Sensitive Calmodulin-Like Protein CALML3 in Normal Human Skin and Hyperproliferative Skin Disorders

Background and Objective

Calmodulin-like protein CALML3 is an epithelial-specific protein regulated during keratinocyte differentiation in vitro. CALML3 expression is downregulated in breast cancers and transformed cell lines making it an attractive marker for tumor formation. The objective of this study was to survey CALML3 localization in normal epidermis and in hyperproliferative skin diseases including actinic keratosis, squamous and basal cell carcinoma as well as verruca and psoriasis and to compare CALML3 immunoreactivity with the proliferation marker Ki-67.

Methods

Paraffin-embedded tissue sections from normal human skin and hyperproliferative skin disorders were examined by immunohistochemistry and analyzed for localization and expression of CALML3 and Ki-67.

Results

CALML3 was strongly expressed in differentiating layers of normal skin, staining the periphery in suprabasal cells and exhibiting nuclear localization in the stratum granulosum. CALML3 nuclear localization was inversely correlated to Ki-67 staining in each disease, indicating that CALML3 nuclear presence is related to terminal cell differentiation and postmitotic state.

Conclusions

Increased CALML3 expression in suprabasal layers is characteristic for differentiating keratinocytes in normal epidermis, and nuclear expression of CALML3 inversely correlates with expression of the proliferation marker Ki-67. This suggests that CALML3 is a useful marker for normal and benign hyperplastic epidermal development, whereas the loss of nuclear CALML3 indicates progression to a proliferative and potentially malignant phenotype.     

207-nm UV Light - A Promising Tool for Safe Low-Cost Reduction of Surgical Site Infections. I: In Vitro Studies

Background

0.5% to 10% of clean surgeries result in surgical-site infections, and attempts to reduce this rate have had limited success. Germicidal UV lamps, with a broad wavelength spectrum from 200 to 400 nm are an effective bactericidal option against drug-resistant and drug-sensitive bacteria, but represent a health hazard to patient and staff. By contrast, because of its limited penetration, ∼200 nm far-UVC light is predicted to be effective in killing bacteria, but without the human health hazards to skin and eyes associated with conventional germicidal UV exposure.

Aims

The aim of this work was to test the biophysically-based hypothesis that ∼200 nm UV light is significantly cytotoxic to bacteria, but minimally cytotoxic or mutagenic to human cells either isolated or within tissues.

Methods

A Kr-Br excimer lamp was used, which produces 207-nm UV light, with a filter to remove higher-wavelength components. Comparisons were made with results from a conventional broad spectrum 254-nm UV germicidal lamp. First, cell inactivation vs. UV fluence data were generated for methicillin-resistant S. aureus (MRSA) bacteria and also for normal human fibroblasts. Second, yields of the main UV-associated pre-mutagenic DNA lesions (cyclobutane pyrimidine dimers and 6-4 photoproducts) were measured, for both UV radiations incident on 3-D human skin tissue.

Results

We found that 207-nm UV light kills MRSA efficiently but, unlike conventional germicidal UV lamps, produces little cell killing in human cells. In a 3-D human skin model, 207-nm UV light produced almost no pre-mutagenic UV-associated DNA lesions, in contrast to significant yields induced by a conventional germicidal UV lamp.

Conclusions

As predicted based on biophysical considerations, 207-nm light kills bacteria efficiently but does not appear to be significantly cytotoxic or mutagenic to human cells. Used appropriately, 207-nm light may have the potential for safely and inexpensively reducing surgical-site infection rates, including those of drug-resistant origin.     

Designed Coiled-Coil Peptides Inhibit the Type Three Secretion System of Enteropathogenic Escherichia coli

Background

Enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC) are two categories of E. coli strains associated with human disease. A major virulence factor of both pathotypes is the expression of a type three secretion system (TTSS), responsible for their ability to adhere to gut mucosa causing a characteristic attaching and effacing lesion (A/E). The TTSS translocates effector proteins directly into the host cell that subvert mammalian cell biochemistry.

Methods/Principal Findings

We examined synthetic peptides designed to inhibit the TTSS. CoilA and CoilB peptides, both representing coiled-coil regions of the translocator protein EspA, and CoilD peptide, corresponding to a coiled–coil region of the needle protein EscF, were effective in inhibiting the TTSS dependent hemolysis of red blood cells by the EPEC E2348/69 strain. CoilA and CoilB peptides also reduced the formation of actin pedestals by the same strain in HEp-2 cells and impaired the TTSS-mediated protein translocation into the epithelial cell. Interestingly, CoilA and CoilB were able to block EspA assembly, destabilizing the TTSS and thereby Tir translocation. This blockage of EspA polymerization by CoilA or CoilB peptides, also inhibited the correct delivery of EspB and EspD as detected by immunoblotting. Interestingly, electron microscopy of bacteria incubated with the CoilA peptide showed a reduction of the length of EspA filaments.

Conclusions

Our data indicate that coiled-coil peptides can prevent the assembly and thus the functionality of the TTSS apparatus and suggest that these peptides could provide an attractive tool to block EPEC and EHEC pathogenesis.     

LF-15 & T7, Synthetic Peptides Derived from Tumstatin,Attenuate Aspects of Airway Remodelling in a Murine Model of Chronic OVA-Induced Allergic Airway Disease

Background

Tumstatin is a segment of the collagen-IV protein that is markedly reduced in the airways of asthmatics. Tumstatin can play an important role in the development of airway remodelling associated with asthma due to its anti-angiogenic properties. This study assessed the anti-angiogenic properties of smaller peptides derived from tumstatin, which contain the interface tumstatin uses to interact with the αVβ3 integrin.

Methods

Primary human lung endothelial cells were exposed to the LF-15, T3 and T7 tumstatin-derived peptides and assessed for cell viability and tube formation in vitro. The impact of the anti-angiogenic properties on airways hyperresponsiveness (AHR) was then examined using a murine model of chronic OVA-induced allergic airways disease.

Results

The LF-15 and T7 peptides significantly reduced endothelial cell viability and attenuated tube formation in vitro. Mice exposed to OVA+ LF-15 or OVA+T7 also had reduced total lung vascularity and AHR was attenuated compared to mice exposed to OVA alone. T3 peptides reduced cell viability but had no effect on any other parameters.

Conclusion

The LF-15 and T7 peptides may be appropriate candidates for use as novel pharmacotherapies due to their small size and anti-angiogenic properties observed in vitro and in vivo.     

Expression of Nucleophosmin/NPM1 correlates with migration and invasiveness of colon cancer cells

Background

We aimed to examine the expression level of Nucleophosmin (NPM1) protein in colon cancer tissues and to investigate the potential role of NPM1 in the regulation of cell migration and invasiveness.

Methods

Immunohistochemical assay was performed to examine the expression pattern of NPM1 in 31 groups of colonic carcinoma samples, including colon tumors, adjacent normal tissues, and matched metastatic lymph nodes from the same patients. Small interfering RNA technique and exogenous expression of wild type NPM1 methods were used to further verify the function of NPM1.

Results

High-expression of NPM1 correlates with lymph node metastasis (P = 0.0003) and poor survival rate of human colon cancer patients (P = 0.017). SiRNA-mediated reduction of NPM1 was also shown to inhibit the migration and invasiveness of metastatic colon cancer HCT116 cell line. In addition, the exogenous expression of NPM1 in HT29 cells, a NPM1 low expression and low invasive colon cancer cell line, enhanced cell migration and invasiveness along with increased cell proliferation.

Conclusions

The current study uncovered the critical role of NPM1 in the regulation of colon cancer cells migration and invasion, and NPM1 may serve as a potential marker for the prognosis of colon cancer patients.     

Mechanisms of GOLPH3 Associated with the Progression of Gastric Cancer: A Preliminary Study

Study Design

To investigate the specific mechanisms by which Golgi phosphoprotein 3 (GOLPH3) affects the progression of gastric cancer and to explore its clinical significance.

Methods

Immunohistochemical analysis was used to evaluate the correlations between GOLPH3, phosphorylated mTOR (p-mTOR), phosphorylated Akt (p-Akt), phosphorylated p70S6 (p-p70S6), phosphorylated 4E-BP1 (p-4E-BP1) and the clinicopathological features of gastric cancer. The mRNA expression levels of GOLPH3, mTOR, Akt, p70S6 and 4E-BP1 in gastric cancer, carcinoma-adjacent and paired normal tissue were analyzed using RT-PCR. Western blotting was used to determine the protein expression of GOLPH3, p-mTOR, p-Akt, p-p70S6 and p-4E-BP1 in tissues.

Results

High expression protein levels of GOLPH3, p-AKT, p-mTOR, p70S6, p-4E-BP1 were positively associated with histological grade (p<0.05), depth of invasion (p<0.05), distant metastasis (p<0.05) and lymph node involvement (p<0.05). Compared with carcinoma-adjacent and paired normal tissues, the mRNA expression levels of GOLPH3, AKT, mTOR, p70S6 and 4EBP1 in gastric cancer tissues were significantly higher. The protein expression levels of GOLPH3, p-AKT, p-mTOR, p-p70S6 and p-4E-BP1 in gastric cancer tissues were also significantly higher than in carcinoma-adjacent and paired normal tissues. A strong positive correlation was observed between GOLPH3, p-mTOR, p-p70S6 and p-4EBP1 expression (r = 0.410, 0.303 and 0.276, respectively, p<0.05), but no significant correlation between the expression of GOLPH3 and p-Akt was observed.

Conclusions

The GOPLH3 expression level is highly correlated with Akt/mTOR signaling in human gastric cancer samples. GOLPH3 combined with Akt/mTOR signaling activation may play an important role in the development, differentiation, invasion and metastasis of gastric cancer.     

MiR-20a Is Upregulated in Anaplastic Thyroid Cancer and Targets LIMK1

Background

There have been conflicting reports regarding the function of miR-20a in a variety of cancer types and we previously found it to be dysregulated in sporadic versus familial papillary thyroid cancer. In this study, we studied the expression of miR-20a in normal, benign and malignant thyroid samples, and its effect on thyroid cancer cells in vitro and in vivo.

Methodology/Principal Findings

The expression of miR-20a in normal, benign and malignant thyroid tissue was determined by quantitative RT-PCR. Thyroid cancer cells were transfected with miR-20a and the effect on cellular proliferation, tumor spheroid formation, and invasion was evaluated. Target genes of miR-20 were determined by genome-wide mRNA expression analysis with miR-20a overexpression in thyroid cancer cells and target prediction database. Target genes were validated by quantitative PCR and immunoblotting, and luciferase assays. MiR-20a expression was significantly higher in anaplastic thyroid cancer than in differentiated thyroid cancer, and benign and normal thyroid tissues. MiR-20a significantly inhibited thyroid cancer cell proliferation in vitro (p<0.01) and in vivo (p<0.01), tumor spheroid formation (p<0.05) and invasion (p<0.05) in multiple thyroid cancer cell lines. We found that LIMK1 was a target of miR-20a in thyroid cancer cell lines and direct knockdown of LIMK1 recapitulated the effect of miR-20a in thyroid cancer cells.

Conclusions/Significance

To our knowledge, this is the first study to demonstrate that miR-20a plays a role as a tumor suppressor in thyroid cancer cells and targets LIMK1. Our findings suggest the upregulated expression of miR-20a in anaplastic thyroid cancer counteracts thyroid cancer progression and may have therapeutic potential.     

Effects of karanjin on cell cycle arrest and apoptosis in human A549, HepG2 and HL-60 cancer cells

Background

We have investigated the potential anticancer effects of karanjin, a principal furanoflavonol constituent of the Chinese medicine Fordia cauliflora, using cytotoxic assay, cell cycle arrest, and induction of apoptosis in three human cancer cell lines (A549, HepG2 and HL-60 cells).

Results

MTT cytotoxic assay showed that karanjin could inhibit the proliferation and viability of all three cancer cells. The induction of cell cycle arrest was observed via a PI (propidium iodide)/RNase Staining Buffer detection kit and analyzed by flow cytometry: karanjin could dose-dependently induce cell cycle arrest at G2/M phase in the three cell lines. Cell apoptosis was assessed by Annexin V-FITC/PI staining: all three cancer cells treated with karanjin exhibited significantly increased apoptotic rates, especially in the percentage of late apoptosis cells.

Conclusion

Karanjin can induce cancer cell death through cell cycle arrest and enhance apoptosis. This compound may be effective clinically for cancer pharmacotherapy.     

Mesenchymal Stem Cells Promote Mammosphere Formation and Decrease E-Cadherin in Normal and Malignant Breast Cells

Introduction

Normal and malignant breast tissue contains a rare population of multi-potent cells with the capacity to self-renew, referred to as stem cells, or tumor initiating cells (TIC). These cells can be enriched by growth as “mammospheres” in three-dimensional cultures.

Objective

We tested the hypothesis that human bone-marrow derived mesenchymal stem cells (MSC), which are known to support tumor growth and metastasis, increase mammosphere formation.

Results

We found that MSC increased human mammary epithelial cell (HMEC) mammosphere formation in a dose-dependent manner. A similar increase in sphere formation was seen in human inflammatory (SUM149) and non-inflammatory breast cancer cell lines (MCF-7) but not in primary inflammatory breast cancer cells (MDA-IBC-3). We determined that increased mammosphere formation can be mediated by secreted factors as MSC conditioned media from MSC spheroids significantly increased HMEC, MCF-7 and SUM149 mammosphere formation by 6.4 to 21-fold. Mammospheres grown in MSC conditioned media had lower levels of the cell adhesion protein, E-cadherin, and increased expression of N-cadherin in SUM149 and HMEC cells, characteristic of a pro-invasive mesenchymal phenotype. Co-injection with MSC in vivo resulted in a reduced latency time to develop detectable MCF-7 and MDA-IBC-3 tumors and increased the growth of MDA-IBC-3 tumors. Furthermore, E-cadherin expression was decreased in MDA-IBC-3 xenografts with co-injection of MSC.

Conclusions

MSC increase the efficiency of primary mammosphere formation in normal and malignant breast cells and decrease E-cadherin expression, a biologic event associated with breast cancer progression and resistance to therapy.     

Cell Surface Protein Disulfide Isomerase Regulates Natriuretic Peptide Generation of Cyclic Guanosine Monophosphate

Rationale

The family of natriuretic peptides (NPs), including atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), and C-type natriuretic peptide (CNP), exert important and diverse actions for cardiovascular and renal homeostasis. The autocrine and paracrine functions of the NPs are primarily mediated through the cellular membrane bound guanylyl cyclase-linked receptors GC-A (NPR-A) and GC-B (NPR-B). As the ligands and receptors each contain disulfide bonds, a regulatory role for the cell surface protein disulfide isomerase (PDI) was investigated.

Objective

We utilized complementary in vitro and in vivo models to determine the potential role of PDI in regulating the ability of the NPs to generate its second messenger, cyclic guanosine monophosphate.

Methods and Results

Inhibition of PDI attenuated the ability of ANP, BNP and CNP to generate cGMP in human mesangial cells (HMCs), human umbilical vein endothelial cells (HUVECs), and human aortic smooth muscle cells (HASMCs), each of which were shown to express PDI. In LLC-PK1 cells, where PDI expression was undetectable by immunoblotting, PDI inhibition had a minimal effect on cGMP generation. Addition of PDI to cultured LLC-PK1 cells increased intracellular cGMP generation mediated by ANP. Inhibition of PDI in vivo attenuated NP-mediated generation of cGMP by ANP. Surface Plasmon Resonance demonstrated modest and differential binding of the natriuretic peptides with immobilized PDI in a cell free system. However, PDI was shown to co-localize on the surface of cells with GC-A and GC-B by co-immunoprecpitation and immunohistochemistry.

Conclusion

These data demonstrate for the first time that cell surface PDI expression and function regulate the capacity of natriuretic peptides to generate cGMP through interaction with their receptors.     

Shikonin enhances efficacy of a gene-based cancer vaccine via induction of RANTES

Background

Shikonin, a phytochemical purified from Lithospermum erythrorhizon, has been shown to confer diverse pharmacological activities, including accelerating granuloma formation, wound healing, anti-inflammation and others, and is explored for immune-modifier activities for vaccination in this study. Transdermal gene-based vaccine is an attractive approach for delivery of DNA transgenes encoding specific tumor antigens to host skin tissues. Skin dendritic cells (DCs), a potent antigen-presenting cell type, is known to play a critical role in transmitting and orchestrating tumor antigen-specific immunities against cancers. The present study hence employs these various components for experimentation.

Method

The mRNA and protein expression of RANTES were detected by RT-PCR and ELISA, respectively. The regional expression of RANTES and tissue damage in test skin were evaluated via immunohistochemistry assay. Fluorescein isothiocyanate sensitization assay was performed to trace the trafficking of DCs from the skin vaccination site to draining lymph nodes. Adjuvantic effect of shikonin on gene gun-delivered human gp100 (hgp100) DNA cancer vaccine was studied in a human gp100-transfected B16 (B16/hgp100) tumor model.

Results

Among various phytochemicals tested, shikonin induced the highest level of expression of RANTES in normal skin tissues. In comparison, mouse RANTES cDNA gene transfection induced a higher level of mRANTES expression for a longer period, but caused more extensive skin damage. Topical application of shikonin onto the immunization site before gene gun-mediated vaccination augmented the population of skin DCs migrating into the draining lymph nodes. A hgp100 cDNA gene vaccination regimen with shikonin pretreatment as an adjuvant in a B16/hgp100 tumor model increased cytotoxic T lymphocyte activities in splenocytes and lymph node cells on target tumor cells.

Conclusion

Together, our findings suggest that shikonin can effectively enhance anti-tumor potency of a gene-based cancer vaccine via the induction of RANTES expression at the skin immunization site.     

Activation of c-Met and Upregulation of CD44 Expression Are Associated with the Metastatic Phenotype in the Colorectal Cancer Liver Metastasis Model

Background

Liver metastasis is the most common cause of death in patients with colorectal cancer. Despite extensive research into the biology of cancer progression, the molecular mechanisms that drive colorectal cancer metastasis are not well characterized.

Methods

HT29 LM1, HT29 LM2, HT29 LM3 cell lines were derived from the human colorectal cancer cell line HT29 following multiple rounds of in vivo selection in immunodeficient mice.

Results

CD44 expression, a transmembrane glycoprotein involved in cell-cell and cell-matrix adhesions, and cancer cells adhesion to endothelial cells was increased in all in vivo selected cell lines, with maximum CD44 expression and cancer cells adhesion to endothelial cells in the highly metastatic HT29 LM3 cell line. Activation of c-Met upon hepatocyte growth factor (HGF) stimulation in the in vivo selected cell lines is CD44 independent. In vitro separation of CD44 high and low expression cells from HT29 LM3 cell line with FACS sorting confirmed that c-Met activation is CD44 independent upon hepatocyte growth factor stimulation. Furthermore, in vivo evaluation of CD44 low and high expressing HT29 LM3 cells demonstrated no difference in liver metastasis penetrance.

Conclusions

Taken together, our findings indicate that the aggressive metastatic phenotype of in vivo selected cell lines is associated with overexpression of CD44 and activation of c-MET. We demonstrate that c-Met activation is CD44 independent upon hepatocyte growth factor stimulation and confirm that CD44 expression in HT29 LM3 cell line is not responsible for the increase in metastatic penetrance in HT29 LM3 cell line.