Vimentin, a major constituent of the intermediate filament (IF) family of proteins, is ubiquitously expressed in normal mesenchymal cells and is known to maintain cellular integrity and provide resistance against stress. Increased vimentin expression has been reported in various epithelial cancers including prostate cancer, gastrointestinal tumors, CNS tumors, breast cancer, malignant melanoma, lung cancer and other types of cancers. Vimentin's over-expression in cancer correlates well with increased tumor growth, invasion and poor prognosis; however, the role of vimentin in cancer progression remains obscure. In the recent years, vimentin has gained much importance as a marker for epithelial-mesenchymal transition (EMT). Although EMT is associated with a number of tumorigenic events, the role of vimentin in the underlying events mediating these processes remains unknown. Though majority of the literature findings indicate a future significance of vimentin as a biomarker for different cancers with clinical relevance, more research in to the molecular aspects will be crucial to particularly evaluate the function of vimentin in the process of tumorigenesis. By virtue of its over-expression in a large number of cancers and its role in mediating various tumorigenic events, vimentin serves as an attractive target for cancer therapy. Further, research directed toward elucidating the role of vimentin in various signaling pathways would open up new approaches for the development of promising therapeutic agents. This review summarizes the expression and functions of vimentin in cancers and also suggests some directions toward future cancer therapy utilizing vimentin as a potential target.
This study’s goal was to assess the safety of tumor-targeted interleukin-12 (ttIL-12) when administered by electrogenetherapy in C3H/HeJ mice by identifying an initial safe dose for human dose escalation schemes, toxicity target organs, markers of toxicity, and toxicity reversibility. Tumor-free mice receiving two doses of 0.45% NaCl, 1 µg ttIL-12 DNA in 0.45% NaCl, or 5 µg ttIL-12 DNA in 0.45% NaCl ten days apart combined with low-intensity electroporation were compared to non-treatment controls over time. All mice had blood cell counts, serum chemistry profiles, plasma IL-12 and IFNγ determinations, necropsy, and multi-organ histopathology. Mild treatment-associated changes included electroporation-associated muscle changes that resolved by 30 days; decreased total white blood cell counts and infectious disease in the 5 µg ttIL-12 group, but not in the 1 µg group; and liver changes in ttIL-12 groups that correlated with alanine transaminase (ALT) levels and resolved by 30 days. Dystrophic cardiac calcification seen in older, 5 µg ttIL-12-treated mice was the only serious toxicity. Based on these results and the lack of any effect on wound healing when combined with surgery, low-intensity electrogenetherapy with ttIL-12 appears to be safe and well tolerated.
Gene therapy; IL-12; tumor targeting; toxicity; cancer; electroporation
Established cell transfection via nucleofection relies on nucleofection buffers with unknown and proprietary makeup due to trade secrecy, inhibiting the possibility of using this otherwise effective method for developing cell therapy. We devised a three-step method for discovering an optimal formulation for the nucleofection of any cell-line. These steps include the selection of the best nucleofection program and known buffer type, selection of the best polymer for boosting the transfection efficiency of the best buffer, and the comparison with the optimal buffer from an established commercial vendor (Amaxa). Using this 3-step selection system, competitive nucleofection formulations were discovered for multiple cell lines, which are equal to or surpass the efficiency of the Amaxa nucleofector solution in a variety of cells and cell lines, including primary adipose stem cells, muscle cells, tumor cells, and immune cells. Through the use of scanning electron microscopy, we have revealed morphological changes, which predispose for the ability of these buffers to assist in transferring plasmid DNA into the nuclear space. Our formulation may greatly reduce the cost of electroporation study in laboratory and boosts the potential of application of electroporation-based cell therapies in clinical trials.
electroporation; cell transfection; cell therapy; adipose stem cells; formulation
Adipose stem cells have a strong potential for use in cell-based therapy, but the current nucleofection technique, which relies on unknown buffers, prevents their use.
We developed an optimal nucleofection formulation for human adipose stem cells by using a three-step method that we had developed previously. This method was designed to determine the optimal formulation for nucleofection that was capable of meeting or surpassing the established commercial buffer (Amaxa), in particular for murine adipose stem cells. By using this same buffer, we determined that the same formulation yields optimal transfection efficiency in human mesenchymal stem cells.
Our findings suggest that transfection efficiency in human stem cells can be boosted with proper formulation.
Electroporation; Formulation; Stem cells; Transfection; Cell therapy
Tumor-targeted antibody therapy is one of the safest biological therapeutics for cancer patients, but it is often ineffective at inducing direct tumor cell death and is ineffective against resistant tumor cells. Currently, the antitumor efficacy of antibody therapy is primarily achieved by inducing indirect tumor cell death, such as antibody-dependent cell cytotoxicity. Our study reveals that Herceptin conjugates, if generated via the crosslinker EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride), are capable of engendering human epidermal growth factor receptor 2 (Her2) positive tumor cells death. Using a high-performance liquid chromatography (HPLC) system, three peaks with estimated molecular weights of antibody monomer, dimer, and trimer were isolated. Both Herceptin trimer and dimer separated by HPLC induced significant levels of necrotic tumor cell death, although the trimer was more effective than the dimer. Notably, the Herceptin trimer also induced Herceptin-resistant tumor cell death. Surprisingly different from the known cell death mechanism that often results from antibody treatment, the Herceptin trimer elicited effective and direct tumor cell death via a novel mechanism: programmed cell necrosis. In Her2-positive cells, inhibition of necrosis pathways significantly reversed Herceptin trimer-induced cell death. In summary, the Herceptin trimer reported herein harbors great potential for overcoming tumor cell resistance to Herceptin treatment.
Based on superior outcomes from electrochemogene therapy (ECGT) compared to electrochemotherapy (ECT)in mice, we determined the efficacy of ECGT applied to spontaneous canine neoplasms. Intralesional bleomycin (BLM) and feline interleukin-12 DNA (fIL-12 DNA) injection combined with trans-lesional electroporation resulted in complete cure of two recurrent World Health Organization (WHO) stageT2bN0M0 oral squamous cell carcinomas (SCCs)and one T2N0M0acanthomatous ameloblastoma. Three remaining dogs, which had no other treatment options, had partial responses to ECGT; one had mandibular T3bN2bM1 melanoma with pulmonary and lymph node metastases; one had cubital T3N0M1 histiocytic sarcoma with spleen metastases; and one had soft palate T3N0M0fibrosarcoma. The melanoma dog had decrease in the size of the primary tumor before recrudescence and euthanasia. The histiocytic sarcoma dog had resolution of the primary tumor, but was euthanized because of metastases four months after the only treatment. The dog with T 3N0M0 fibrosarcoma had tumor regression with recrudescence. Treatment was associated with minimal side effects and was easy to perform; was associated with repair of bone lysis in cured dogs; improved quality of life for dogs with partial responses; and extended overall survival time. ECGT appears to be a safe and resulted in complete responses in SCC and acanthomatous ameloblastoma.
Electroporation; Bleomycin; IL-12; Canine; Neoplasia; Gene therapy
Crosstalk between tumor cells and the cognate microenvironment plays a crucial role in tumor initiation and progression. However, only a few genes are known to affect such a crosstalk. This study reveals that WSX1 plays such a role when highly expressed in tumor cells. The expression of WSX1 in Lewis Lung Carcinoma (LLC) and the melanoma cell line AGS induces the death of T cells and inhibits the production of the effector cytokine IFNγ from NK and T cells, resulting in the promotion of tumor growth. These pro-tumorigenic properties of WSX1 are independent of IL27. This key observation reveals a new pathway of tumor-host interaction, which will ultimately lead to better strategies in immune therapy to reverse tumor tolerance.
Eradication of residual malignancies and metastatic tumors via a systemic approach is the key for successfully treating cancer and increasing the cancer patient survival. Systemic administration of IL12 protein in an acute large dose is effective but toxic. Systemic administration of IL12 gene by persistently expressing a low level of IL12 protein may reduce the systemic toxicity, but only eradicates IL12 sensitive tumors. Here, we discovered that sequential administration of IL12 and IL27 encoding DNA, referred to as sequential IL12-IL27 gene therapy, not only eradicated IL12 sensitive tumors from 100% of mice but also eradicated the highly malignant 4T1 tumors from 33% of treated mice in multiple independent experiments. This IL12-IL27 sequential gene therapy is not only superior to IL12-IL12 sequential gene therapy for eliminating tumors, but also for inducing CTL activity, increasing T cell infiltration into tumors, and yielding a large number of tumor-specific IFNγ positive CD8 T cells. Notably, depletion of either T- or NK-cells during the IL27 treatment phase reverses tumor eradication, suggesting an NK-cell requirement for this sequential gene therapy-mediated tumor eradication. Both reversal of the administration sequence and co-administration of IL12 and IL27 impaired the tumor eradication in 4T1 tumor bearing mice. This IL12-IL27 sequential gene therapy, via sequential administration of IL12 and IL27 encoding plasmid DNA into tumor-bearing mice through intramuscular electroporation, provides a simple but effective approach for eliminating inaccessible residual tumors.
IL12; IL27; electroporation; gene therapy; tumors; T cells
Electroporation; Gene Delivery; Gene Therapy; DNA vaccine; large animals; Gene expression
Two important aspects for gene therapy are to increase the level of gene expression and track the gene delivery site and expression, and a sensitive reporter gene may be one of the options for preclinical studies and possibly for human clinical trials. We report the novel concept of increasing the activity of the gene products. With the insertion of the mini-peptide-coding sequence CWDDWLC into the plasmid DNA of a SEAP reporter gene, we observed vast increases in the enzyme activity in vitro in all murine and human cell lines used. Also, in vivo injection of this CWDDWLC-SEAP encoding gene resulted in the same increases in reporter gene activity, but these increases did not correspond to alterations in the level of the gene products in the serum. Minor sequence changes in this mini-peptide negate the activity increase of the reporter gene. We report the novel concept of increasing the activity of gene products as another method to improve the reporting sensitivity of reporter genes. This improved reporter gene could complement any improved vector for maximizing the reporter sensitivity. Also, this strategy has the potential to be used to discover peptides that improve the activity of therapeutic genes.
Reporter gene; SEAP; peptide; vector; in vivo
It is well known that the interleukin (IL) 27 receptor WSX1 is expressed in immune cells and induces an IL27-dependent immune response. Opposing this conventional dogma, this study reveals a much higher level of WSX1 expression in multiple types of epithelial tumor cells when compared to normal epithelial cells. Expression of exogenous WSX1 in epithelial tumor cells suppresses tumorigenecity in vitro and inhibits tumor growth in vivo. Different from the role of WSX1 in immune cells, the antitumor activity of WSX1 in epithelial tumor cells is independent of IL27 signaling but is mainly dependent on NK cell surveillance. Deficiency of either the IL27 subunit Epstein–Barr virus-induced gene 3 (EBI3) or the IL27 receptor WSX1 in the host animals had no effect on tumor growth inhibition induced by WSX1 expression in tumor cells. Expression of WSX1 in epithelial tumor cells enhances NK cell cytolytic activity against tumor cells, while the absence of functional NK cells impairs the WSX1-mediated inhibition of epithelial tumor growth. The underlying mechanism by which WSX1 expression in tumor cells enhances NK cytolytic activity is dependent on upregulation of NKG2D ligand expression. Our results reveal an IL27-independent function of WSX1—sensitizing NK cell-mediated antitumor surveillance via an NKG2D-dependent mechanism.
Innate Immunity; NK cell; IL27; Interleukin; TCCR; Tumor; WSX1
Premature termination codons (PTCs) are known to decrease mRNA levels. Here, we report our investigation of the mechanism for this downregulation using the TCR-β gene, which acquires PTCs as a result of programmed rearrangements that occur during normal thymic development. We found that a mini-gene version of this gene, which contains only three TCR-β exons, exhibited efficient downregulation in response to PTCs. This demonstrates that the full coding sequence is not necessary for appropriate regulation. Mutation of the translation start AUG and a downstream in-frame AUG that displayed similarity to the Kozak consensus sequence reversed the downregulatory response to PTCs. Thus, an AUG start codon is required to define the reading frame of a PTC. Specific suppressor tRNAs also reversed the downregulatory response, strongly implicating the involvement of a translation-like process. Remarkably, the addition of suppressor tRNAs or the inactivation of the start AUGs caused a dramatic rise in the levels of PTC-bearing transcripts in the nuclear fraction prepared by two independent methods. Collectively, our results provide evidence for a codon-based surveillance mechanism associated with the nucleus that downregulates aberrant transcripts encoding potentially toxic polypeptides from nonproductively rearranged genes.