Development of targeted therapy for a broad spectrum of cancers (pancreatic cancer, ovarian cancer, glioblastoma and HCC) mediated by a double promoter plasmid expressing diphtheria toxin under the control of H19 and IGF2-P4 regulatory sequences

Abstract

Background: The human IGF2-P4 and H19 promoters are highly active in a variety of human cancers, while existing at a nearly undetectable level in the surrounding normal tissue. Single promoter vectors expressing diphtheria toxin A-fragment (DTA) under the control regulation of IGF2-P4 or H19 regulatory sequences (IGF2-P4-DTA and H19-DTA) were previously successfully used in cell lines, animal models and recently in human patients with superficial cell carcinoma of the bladder, pancreatic cancer and ovarian cancer (treated with H19-DTA). However this targeted medicine approach may be limited, as not all cancer patients express high levels of H19 and it requires prerequisite diagnostic test for H19. Hence, a double promoter DTA-expressing vector was created, carrying on a single construct two separate genes expressing the diphtheria toxin A-fragment (DTA), from two different regulatory sequences, selected from the cancer-specific promoters H19 and IGF2-P4.

Methods: H19 and IGF2-P4 gene expression was tested in cell lines of a broad spectrum of different carcinomas (bladder, pancreas, ovary, glioblastoma and HCC), by RT-PCR. The therapeutic potential of the double promoter toxin vector H19-DTA-(IGF2)-P4-DTA was tested in the different cancer cell lines.

Results: The double promoter vector exhibited superior inhibition activity compared to the single promoter expression vectors, in the different cancer cell lines furthermore, the double promoter vector H19-DTA-P4-DTA exhibited augmented-than-additive anti-cancer activity relative to single promoter expression vectors carrying either DTA sequence alone, when tested in a broad spectrum of tumor cells.

Conclusions: Our findings show that administration of H19-DTA-P4-DTA has the potential to reach tumor cells, deliver its intracellular toxin without targeting normal tissues, and thus may help reduce tumor burden, improve the quality of life of the patient; and prolong their life span. As H19 and IGF2 were expressed in a broad spectrum of different cancers, therefore we propose a double promoter expression approach for treating a variety of tumors expressing H19, IGF2, or both. According to this approach patients may be treated with a single double promoter expression toxin vector which is under the control of the IGF2 and H19 regulatory sequences, differentially expressed in those cancers. As majority of the tumor cells express H19, IGF2, or both, therefore the use of prerequisite diagnostic test will be unnecessary.

Keywords: H19; IGF2; diphtheria toxin A; glioblastoma; hepatocellular carcinoma; ovarian cancer; pancreatic cancer; targeted cancer therapy.

Figure 1

A schematic illustration depicting the construction of the double promoter H19-DTA-P4-DTA expression vector: The coding sequence of each DTA is under the transcriptional control of both H19 and IGF2-P4 promoter sequences, respectively, Kana (R) – kanamycin resistance gene.

Figure 2

The expression of H19 and IGF2 in different cancer cell lines determined by RT-PCR: Shown are RT-PCR analyses of H19 (upper band) and IGF2-P4 (lower band) in human cancer cell lines of (A): Hep3B (hepatocellular carcinoma), ES-2 (ovarian cancer) and CRL-1469 (pancreatic cancer), and in human glioma cell lines (B): A172 ((glioblastoma), U87 (glioblastoma) and GL261 (mouse glioblastoma). ‘M’, 100bp DNA ladder, ‘C’, negative control. Lower figures, are RT-PCR product of histone internal control.

Figure 3

The enhanced activity of H19-DTA-P4-DTA vector in a spectrum of tumor cells: Protein synthesis inhibition activity of the H19-DTA (white), P4-DTA (gray) and H19-DTA-P4-DTA (black) vectors in Hep3B (A), ES-2 (B), CRL-1469 (C), U87 (D) and A172 (E) cells, was measured as a reduction of LucSV40 activity. Cells were co-transfected with 2μg of LucSV40, and with different concentrations (0.005mg - 0.05mg/well) of the DTA expressing vectors or with LucSV40 alone. Transfection experiments were stopped after 48 hours and luciferase activity was assessed. The decrease in LucSV40 activity was determined by comparison to the same cell type transfected with LucSV40 alone as a measure for cytotoxicity. The diverse effect of each vector at the lowest plasmid transfected concentration (0.005mg) is indicated.

Figure 4

Augmented-than-additive activity of H19-DTA-P4-DTA in human different cancer cell lines: The additive effect of H19-DTA-P4-DTA vector was tested in Hep3B cells (A), ES-2 (B), CRL-1469 (C) and U87(D) cells transfected with only 0.005mg H19-DTA-P4-DTA (black) and compared to combination transfection of both vectors (white) H19-DTA + P4-DTA (which the total transfected concentration was therefore twice (0.005mg + 0.005mg).