Enhancement of anti-melanoma activity of a plasmid expressing HIV-1 Vpr delivered through in vivo electroporation

Abstract

The development of novel treatment strategies for the effective delivery of new therapies directed against solid tumors, particularly metastatic melanoma, are required. A novel therapeutic property has previously been discovered for the HIV-1 accessory protein viral protein R (Vpr), based on the ability of this protein to induce G(2) cell cycle arrest as well as apoptosis in various tumor cell lines. Likewise, in vivo electroporation has been utilized as an effective delivery platform for DNA plasmids expressing potentially therapeutic proteins and has been targeted to normal tissues as well as tumors. Our previous findings demonstrated that delivery of a Vpr expression plasmid (pVpr) to established subcutaneous B16.F10 melanoma tumors by in vivo electroporation yielded long-term complete tumor regression in a small percentage of mice. In this study, we modified the electroporation regimen for pVpr with the goal of enhancing the anti-tumor activity of Vpr. pVpr was injected intratumorally, on days 0, 2 and 4, into established subcutaneous B16.F10 melanoma tumors followed by in vivo electroporation. Treatment with 100 mg of pVpr plus electroporation on the modified treatment days resulted in 50% of the mice undergoing complete tumor regressions coupled with long-term survival (i.e., greater than 100 days post treatment). Additional investigations established the intratumoral expression of Vpr and induction of apoptosis for a period of at least seven days after the modified pVpr treatment regimen. This report demonstrates that the anti-tumor activity of a pVpr plus electroporation regimen against established subcutaneous B16.F10 melanoma tumors can be significantly enhanced by a modified treatment schedule. In addition, it appeared that this enhanced anti-tumor effect was correlated with prolonged Vpr expression and the associated induction of intratumoral apoptosis.

Anti-tumor activity mediated by protein and peptide transduction of HIV viral protein R (Vpr)

“…The DNA based reagents were, as indicated, either delivered through live viral vectors or naked DNA plasmids via in vivo electroporation. 9,12,13,15,59,60 As indicated, these experiments demonstrated some anti cancer activity for Vpr, albeit at a low to moderate level. Therefore, we reasoned that other investigations were warranted to evaluate other Vpr based reagents (i.e., peptides or PTD conjugated protein) in terms of potential anticancer activity.…”

“…F10 murine melanoma tumors. 12,13 In these experiments, a small but statistically significant percentage of mice underwent complete regression of their established subcutaneous tumors after treatment. This tumor regression effect is thought to be biologically significant, and potentially applicable to human tumors, since the B16.F10 carboxy region Vpr peptides spanning amino acids 65 through 91.…”

“…[3][4][5][6][7] In addition, Vpr has been demonstrated to inhibit in vitro tumor cell growth of several varied tumor lines, including those of human origin. [8][9][10][11][12][13][14] Also, other studies have demonstrated that this antitumor activity of Vpr is independent of the p53 expression status of the tumor cells. 15 It has been further indicated that Vpr appears to preferentially inhibit the growth of rapidly dividing and transformed cells as opposed to slowly replicating non-transformed cells which further suggests that this protein may have a useful therapeutic utility against specific cancers without significant toxicity against normal cells.…”

“…This is relevant since the Vpr delivery systems employed to date, i.e., through intratumoral electroporation of naked DNA plasmids or virally based expression vectors, have been somewhat inefficient which could account for the minimal to moderate therapeutic index currently observed with these reagents. 12,13,15 …”

The HIV-Derived Protein Vpr52-96 Has Antiglioma Activity In Vitro and In Vivo

“…Various tumor entities are sensitive to Vpr, including neuroblastoma (LAN-2), lymphoma (U937), WHO grade III astrocytoma (U373), cervical cancer (HeLa), liver (HepG2), kidney (293T), melanoma (B16.F10) and leukemia (Jurkat T) cells [8,[20][21][22]. Consequently, first successful approaches to explore the therapeutic efficacy of Vpr were made in gene transfer studies, where Vpr overexpression inhibited growth of melanoma (B78/H1) and oral squamous cell carcinoma cell lines (AT-84) in vitro and in vivo [10,23,24]. The protein was also already employed as a local therapeutic agent by Siddiqui and colleagues, who injected Vpr into mammary carcinoma allografts in mice and observed efficient tumor regression with development of central necrosis in Vpr-treated tumors [18].…”