Suppression of hepatoma tumor growth by systemic administration of the phytotoxin gelonin driven by the survivin promoter.

UNLABELLED: Hepatocellular carcinoma (HCC) is one of the most common types of cancer worldwide. However, there is currently no effective therapy strategy in the clinical practice. Recombinant phytotoxin gelonin fused to other factors have been used to treat different cancers. But there have been no reports of gelonin gene therapy. In this study, we have constructed a recombinant plasmid which contained a tumor-specific survivin promoter to drive phytotoxin gelonin (pSur-Gel). And the cytotoxicity effects of pSur-Gel in HCC were also validated both in vitro and in vivo. The expression level of survivin was detected in different liver cancer cell lines and normal liver cell lines by western blot analysis, and a survivin promoter-driven green fluorescent protein (GFP) expression vectors (pSur-GFP) was also tested in liver cancer cell line HepG2 and normal liver cell line LO2. Moreover, phytotoxin gelonin expression experiment and cytotoxicity experiment of pSur-Gel was performed in HepG2 cells and LO2 cells in vitro. Furthermore, anti-tumor effect of pSur-Gel against HepG2 xenografts and toxicity of this gene were evaluated in the mice model. Finally, LDH release assay, apoptosis assay and immunoblot analyse LC3 conversion (LC3-I to LC3-II) were tested. We found that the expression of survivin protein was higher in liver cancer cell lines compared with the normal liver cells. Further study showed that the pSur-GFP and pSur-Gel was expressed specially in liver cancer cell other than in normal liver cells. pSur-Gel plasmid could effectively inhibit the proliferation of liver cancer cells (*P<0.05), and significantly repress the growth of HepG2 xenografts via intravenous in vivo (*P<0.05). Otherwise, compared to cytomegalovirus promoter-driven gelonin expression vectors (pCMV-Gel), no significantly systemic toxicity or organ injuries had been observed in pSur-Gel treated mice. Further studies revealed that the phytotoxin gelonin induced cell death might be mediated by apoptosis and the damage of cell membrane. Taken together, treating hepatocellular carcinoma with the pSur-Gel may be a novel and interesting cancer gene therapy protocol and is worthy of further development for future clinical trials. KEYWORDS: liver cancer, gelonin, survivin promoter, gene therapy.

IB4 afferent sprouting contributes to bladder dysfunction in spinal rats.

We investigated the role that nonpeptidergic isolectin-B4 (IB4) positive, primary afferent sprouting plays in bladder dysfunction after spinal cord transection (SCT). Rats were implanted with an indwelling bladder cannula and subjected to a complete spinal cord transection at T9/T10. In one group of rats IB4-positive terminals increased below the level of the injury in L6 cord in laminae I and III-VI as early as 3 days after transection, and remained increased 8 and 21 days after transection. Growth associated protein 43 (Gap-43) was expressed on IB4-positive neurons 3 days post-transection and the number of L6 dorsal root ganglia (DRG) neurons expressing IB4 did not change after injury. In another set of experiments IB4-saporin or saporin alone was administered intrathecally to L6/S1 cord. IB4-positive afferents sprouted in L6 cord of saporin only treated rats but IB4 afferent labeling was decreased by 42 and 33% in L6 cord and DRG 21 days after IB4-saporin treatment. IB4-saporin treated rats voided with an efficiency of 28.3% 10-14 days after transection whereas one week later voiding efficiency increased to 86.1%. Inefficient voiding by saporin and 10-14 day IB4-saporin treated rats was linked to voiding that occurred after the peak in micturition pressure. On the other hand, increased voiding efficiency in 20-30 day IB4-saporin treated rats was associated with voiding occurring before the peak of the micturition pressure. These results suggest that IB4-positive afferent sprouting plays a role in the generation of bladder dysfunction following SCT.

Selective ablation of GABA neurons in the ventral tegmental area increases spontaneous locomotor activity.

Gamma-aminobutyric acid (GABA) neurons in the ventral tegmental area (VTA) provide innervation to cortical and subcortical regions of the brain. To solidify the importance of these VTA GABA neurons in behavioral function, we employed the neurotoxin dermorphin-saporin (DS) to selectively lesion VTA GABA neurons prior to assessing spontaneous motor activity. Rats were bilaterally microinfused with DS (1.0 or 2.0 pmol/200 nl/side) or blank-saporin control (BS, 200 nl/side) into the VTA. Seven days later, DS-treated rats exhibited significantly elevated motility in comparison with BS-treated rats; this elevated motility normalized by Day 14 following pretreatment with 1.0 pmol of DS but was sustained on Day 14 after pretreatment with 2.0 pmol of DS. A selective loss of VTA GABA neurons on Day 14 was demonstrated through reduced expression of mRNA for glutamic acid decarboxylase-67 and micro-opioid receptor, but not tyrosine hydroxylase (a dopamine neuron marker), in the VTA. Thus, a dose- and time-related selective loss of VTA GABA neurons was accomplished using this novel neurotoxin. This loss of GABA VTA neurons was associated with hypermotility, further supporting their important regulatory role in the generation of behavior.