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. 2009 Aug 5;1(2):85-91.
doi: 10.4255/mcpharmacol.09.10.

HIV Nef-M1 Effects on Colorectal Cancer Growth in Tumor-induced Spleens and Hepatic Metastasis

Willie Harrington  1 , Vincent Bond, Ming Bo Huang, Michael Powell, James Lillard, Upender Manne, Harvey Bumpers
Affiliations

HIV Nef-M1 Effects on Colorectal Cancer Growth in Tumor-induced Spleens and Hepatic Metastasis

Willie Harrington et al. Mol Cell Pharmacol. .

Abstract

CXCR4 receptors have been implicated in tumorigenesis and proliferation, making it a potential target for colorectal cancer therapy. Expression of this chemokine receptor on cellular surfaces appears to promote metastasis by directly stimulating tumor cell migration and invasion. The receptor/ligand, CXCR4/SDF-1alpha, pair are critically important to angiogenesis and vascular remodeling which supports cancer proliferation. Our work has shown that a novel apoptotic peptide of HIV-1, Nef-M1, can act as a CXCR4 antagonist, inducing apoptosis in CXCR4 containing cells. Four colorectal tumor cell lines (HT-29, LS174t, SW480, WiDr), were evaluated for their response to Nef-M1 peptide via in vivo and in vitro. The presence of CXCR4 receptors on tumor cells was determined using immunohistochemical and RT-PCR analyses. Solid xenografts derived from tumor cell lines grown in SCID mice, were evaluated for the persistence of the receptor. Xenografts propagated in SCID mice from each of the four cell lines demonstrated high levels of receptor expression as well. The effects of Nef-M1 in vivo via splenic injected mice and subsequent hepatic metastasis also demonstrated dramatic reduction of primary tumor growth in the spleen and secondary invasion of the liver. We concluded that Nef-M1 peptide, through physical interaction(s) with CXCR4, drives apoptotic reduction in in vivo primary tumor growth and metastasis.

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Figures

Figure 1
Figure 1. CXCR4 protein expression in colorectal cells was analyzed by immunocytochemistry
A. Colorectal cancer cell lines were evaluated for CXCR4 receptor expression. The top row is unstained cells and the bottom is IHC staining for CXCR4 using flourescein isothiocynatae (FITC) green. B. Breast cancer cells, MDA-MB-231 and MDA-MB- 468 were positive and negative controls, respectively. Magnification: 400x.
Figure 2
Figure 2. CXCR4 transcription was analyzed in colorectal tumor cell lines and primary tissue by Reverse Transcription Polymerase Chain Reaction (PCR)
RNA was isolated from cells or from tumor tissue and reverse transcribed. The resultant PCR products were fractionated on a 1.5% agarose gel followed by ethidium bromide staining, and then analyzed by ABI Prism 3130 xI Sequence Detection System and 3130 xI Genetic Analyzer data collection software v 3.0. Lane M: 100 bp DNA ladder; lane 1: 1097 bp CXCR4 band; lane 2: 922 bp CXCR4 band; lane 3: 508 bp CXCR4 band. (I) As a positive control for RNA expression of CXCR4, a breast cancer cell line, MDA-MB-231, and in breast tumor tissue were analyzed. (II) Four colorectal adenocarcinoma cell lines (LS-174T, SW480, HT-29, WiDr) were analyzed for CXCR4 transcription. (III) Tumor xenografts grown subcutaneously from injected cells were also analyzed for CXCR4 transcription. The PCR products were designed to determine whether the CXCR4 transcript has deletions in it (e.g., IIb, WiDr appears to be a truncated transcript).
Figure 3
Figure 3. Primary tumor growth and liver metastasis
Spleens and livers excised from the SCID mice four weeks after spleens were inoculated with tumor cells (A-HT-29, B-WiDr, C-LS174t, D-SW480). Primary tumor growth was seen in the spleens of all mice. All mice were untreated. Pictures were taken using a Kodak DC290 zoom digital camera.
Figure 4
Figure 4. Primary splenic tumors
Splenic tumors of mice treated with Nef-M1 (N), Control (C), and scramble peptide (S) bi-weekly for one month following spleens inoculation with CRC cells. Pictures were taken using a Kodak DC290 zoom digital camera.
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