• Asian Pacific Journal of Cancer Prevention
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• v.14 no.5
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• pp.2789-2800
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• 2013

Background: The aim of this systematic review was to investigate whether stem cells could be effectively applied in targeted therapy of breast cancer. Material and Method: A systematic literature search was performed for original articles published from January 2007 until May 2012. Results: Nine studies met the inclusion criteria for phase I or II clinical trials, of which three used stem cells as vehicles, two trials used autologous hematopoetic stem cells and in four trials cancer stem cells were targeted. Mesenchymal stem cells (MSCs) were applied as cellular vehicles to transfer therapeutic agents. Cell therapy with MSC can successfully target resistant cancers. Cancer stem cells were selectively targeted via a proteasome-dependent suicide gene leading to tumor regression. $Wnt/{\beta}$-catenin signaling pathway has been also evidenced to be an attractive CSC-target. Conclusions: This systematic review focused on two different concepts of stem cells and breast cancer marking a turning point in the trials that applied stem cells as cellular vehicles for targeted delivery therapy as well as CSC-targeted therapies. Applying stem cells as targeted therapy could be an effective therapeutic approach for treatment of breast cancer in the clinic and in therapeutic marketing; however this needs to be confirmed with further clinical investigations.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.12
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• pp.7489-7496
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• 2013

This study was conducted to investigate enhancement of anti-tumor effects of the lentogenic Newcastle disease virus Clone30 strain (NDV rClone30) expressing cytosine deaminase (CD) gene against tumor cells and in murine groin tumor-bearing models. Cytotoxic effects of the rClone30-CD/5-FC on the HepG2 cell line were examined by an MTT method. Anti-tumor activity of rClone30-CD/5-FC was examined in H22 tumor-bearing mice. Compared to the rClone30-CD virus treatment alone, NDV rClone30-CD/5-FC at 0.1 and 1 MOIs exerted significant cytotoxic effects (P<0.05) on HepG2 cells. For treatment of H22 tumor-bearing mice, recombinant NDV was injected together with 5-FC given by either intra-tumor injection or tail vein injection. When 5-FC was administered by intra-tumor injection, survival for the rClone30-CD/5-FC-treated mice was 4/6 for 80 days period vs 1/6, 0/6 and 0/6 for the mice treated with rClone30-CD, 5-FC and saline alone, respectively. When 5-FC was given by tail vein injection, survival for the rClone30-CD/5-FC-treated mice was 3/6 vs 2/6, 0/6 and 0/6 for the mice treated with rClone30-CD, 5-FC or saline alone, respectively. In this study, NDV was used for the first time to deliver the suicide gene for cancer therapy. Incorporation of the CD gene in the lentogenic NDV genome together with 5-FC significantly enhances cell death of HepG2 tumor cells in vitro, decreases tumor volume and increases survival of H22 tumor-bearing mice in vivo.

• Genomics & Informatics
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• v.6 no.2
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• pp.84-86
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• 2008

The Tetrahymena group I intron has been shown to employ a trans-splicing reaction and has been modified to specifically target and replace human telomerase reverse transcriptase (hTERT) RNA with a suicide gene transcript, resulting in the induction of selective cytotoxicity in cancer cells that express the target RNA, in animal models as well as in cell cultures. In this study, we evaluated the target RNA specificity of trans-splicing phenomena by the group I intron in mice that were intraperitoneally inoculated with hTERT-expressing human cancer cells to validate the anti-cancer therapeutic applicability of the group I intron. To this end, an adenoviral vector that encoded for the hTERT-targeting group I intron was constructed and systemically injected into the animal. 5'-end RACE-PCR and sequencing analyses of the trans-spliced cDNA clones revealed that all of the analyzed products in the tumor tissue of the virus-infected mice resulted from reactions that were generated only with the targeted hTERT RNA. This study implies the in vivo target specificity of the trans-splicing group I intron and hence suggests that RNA replacement via a trans-splicing reaction by the group I intron is a potent anti-cancer genetic approach.

• Tuberculosis and Respiratory Diseases
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• v.49 no.3
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• pp.298-309
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• 2000

Background : The antitumor effects of herpes simplex virus thymidine kinase (HSV-tk) and ganciclovir (GCV) strategies for cancer gene therapy have a the following advantages : 1) a direct cytotoxicity to HSV-tk modified cancer cells by GCV 2) a cell death by the local transfer of toxic metabolites from the HSV-tk modified cells to nearby unmodified tumor cells (bystander effect), and 3) in vivo bystander effect such as antitumor-immunity. Retroviral and adenoviral sequences can silence transgene expression in cells and mice. In this study, we investigated the above described advantages of HSV-tk/GCV strategy in Lewis lung cell and mouse lung cancer model using retroviral vector and adenoviral vector. Also, we observed whether the expression of a silenced gene can be reactivated by treating cells with butyrate. Methods : Retrovirus-HSV-tk and adenovirus-HSV-tk vectors were used for the transduction of Lewis lung carcinoma (LLC) cells. The change of HSV-tk expression by butyrate was measured by Western blol The antitumor activities containing bystander effect were observed in vivo (by MTT assay) and in vivo tumor models of various combinations of LLC and LLC-tk. Results : 1. Butyrate induced the enhancement of HSV-tk expression from adenovirally transduced cells but not from retrovirally transduced cells. 2. Both retrovirus-HSV-tk and adenovirus-HSV-tk vectors with GCV treatment were effective for killing of tumor cell in vitro and suppression of LLC tumorigenicity. Bystander effect was responsible for killing of mixture of LLC-tk and LLC in vitro and in vivo-tumorigenicity model. Conclusion : Butyrate could augment adenovirus-mediated HSV -tk gene expression. Cancer gene therapy with HSV-tk suicide gene by retroviral and adenoviral vector seems to be an effective approach for lung cancer therapy.

• Tuberculosis and Respiratory Diseases
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• v.46 no.2
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• pp.229-240
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• 1999

Background: The impact of the immune response on cancer gene therapy using viral vectors to deliver a "suicide gene" is currently unclear. A vigrous immune response targeted at viral proteins or transgene may enhance the efficacy of tumor destruction and even augment responses to tumor antigens. These responses may involve the release of cytokines and stimulation of tumor specific cytotoxic T-lymphocytes that enhance therapeutic efficacy. On the other hand, a vigorous rapid cellular immune response may destroy cells expressing the therapeutic gene and attenuate the response to therapy. Furthermore, development of neutralizing antibody responses may prevent readministration of virus, a potentially significant limitation. Evaluating the significance of these limitations in animal models and developing solutions are therefore of obvious importance. Methods: After retroviral transduction of mouse mesothelioma cell line(AB12) with Herpes Simplex Virus thymidine kinase (HSVtk) gene in vitro, subcutaneous flank tumors were established. To study the effect of intact immune system on efficacy of tumor erradication, the ability of the HSVtk/ganciclovir system to inhibit tumor growth was compared among normal Balb/c mice, immunodeficient Balb/c-nude and SCID mice, and Balb/c mice immunosuppressed with cyclosporin. Results: Ganciclovir treatment resulted in greater inhibition of tumor growth in Balb/c mice compared with immunodeficient Balb/c-nude mice and SCID mice(in immunodeficient mice, there were no growth inhibition by ganciclovir treatment). Ganciclovir treatment resulted in greater inhibition of tumor growth in noncyclosporin (CSA) treated Balb/c mice compared with CSA treated Balb/c mice. On day 8, mean ganciclovir-treated tumor volume were 65% of control tumor volume in Balb/c mice versus 77% control tumor volume in CSA-treated Balb/c mice. This effect was still evident during therapy (day 11 and 13). On day 13, non-CSA treated tumor volume was 35% of control tumor volume versus 60% of control tumor volume in CSA treated Balb/c mice. Duration of expression of HSVtk was not affected by the immunosuppression with CSA. Conclusion: These results indicate that the immune responses against retrovirally transduced cells enhance the efficacy of the HSVtk/ganciclovir system. These findings have important implications for clinical trials using currently available retrovirus vectors as well as for future vector design.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.12
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• pp.5043-5047
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• 2014

Nowadays increasing effectiveness in cancer therapy and investigation of formation of new strategies that enhance antiproliferative activity against target organs has become a subject of interest. Although the molecular mechanisms of apoptosis can not be fully explained, it is known that cell suicide program existing in their memory genetically is activated by pathophysiological conditions and events such as oxidative stress. Low pressure (hypobaric) conditions that create hypoxia promote apoptosis by inhibiting cell cycling. In this study, determination of the effects of fractional hypobaric applications at different times on HeLa cells at cellular and molecular levels were targeted. Experiments were carried out under hypobaric conditions (35.2 kPa) in a specially designed hypobaric cabin including 2% $O_2$ and 98% N. Application of fractional hypobaric conditions was repeated two times for 3 hours with an interval of 24 hours. At the end of the implementation period cells were allowed to incubate for 24 hours for activation of repair mechanisms. Cell kinetic parameters such as growth rate (MTT) and apoptotic index were used in determination of the effect of hypobaric conditions on HeLa cells. Also in our study expression levels of the Bcl-2 gene family that have regulatory roles in apoptosis were determined by the RT-PCR technique to evaluate molecular mechanisms. The results showed that antiproliferative effect of hypobaric conditions on HeLa cells started three hours from the time of application and increased depending on the period of exposure. While there was a significant decrease in growth rate values, there was a significant increase in apoptotic index values (p<0.01). Also molecular studies showed that hypobaric conditions caused a significant increase in expression level of proapoptotic gene Bax and significant decrease in antiapoptotic Bfl-1. Consequently fractional application of hypobaric conditions on HeLa cell cultures increased both antiproliferative and apoptotic effects and these effects were triggered by the Bax gene.

• Biomolecules & Therapeutics
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• v.22 no.2
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• pp.114-121
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• 2014

Refractoriness of acute myeloid leukemia (AML) cells to chemotherapeutics represents a major clinical barrier. Suicide gene therapy for cancer has been attractive but with limited clinical efficacy. In this study, we investigated the potential application of herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) based system to inhibit chemoresistant AML cells. We first generated Ara-C resistant K562 cells and doxorubicin-resistant THP-1 cells. We found that the HSV-TK/GCV anticancer system suppressed drug resistant leukemic cells in culture. Chemoresistant AML cell lines displayed similar sensitivity to HSV-TK/GCV. Moreover, HSV-TK/GCV killing of leukemic cells was augmented to a mild but significant extent by all-trans retinoic acid (ATRA) with concomitant upregulation of Connexin 43, a major component of gap junctions. Interestingly, HSV-TK/GCV killing was enhanced by expression of vesicular stomatitis virus G glycoprotein (VSV-G), a fusogenic membrane protein, which also increased leukemic cell fusion. Co-culture resistant cells expressing HSV-TK and cells stably transduced with VSV-G showed that expression of VSV-G could promote the bystander killing effect of HSV-TK/GCV. Furthermore, combination of HSV-TK/GCV with VSV-G plus ATRA produced more pronounced antileukemia effect. These results suggest that the HSV-TK/GCV system in combination with fusogenic membrane proteins and/or ATRA could provide a strategy to mitigate the chemoresistance of AML.

• The Korean Journal of Nuclear Medicine Technology
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• v.15 no.2
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• pp.72-75
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• 2011

Purpose: The HSV1-tk reporter gene system is the most widely used system because of its advantage is that it is possible to monitor directly without the introduction of a separate reporter gene in case of HSV1-tk suicide gene therapy. This study was performed to automate 9-(4-[$^{18}F$] Fluoro-3-hydroxymethylbutyl) guanine ([$^{18}F$] FHBG) that are widely used as substrate for the HSV1-tk reporter gene in living organisms with positron emission tomography (PET) and find the optimized conditions of synthesis. Materials and Methods: Fully automated synthesis of [$^{18}F$] FHBG was performed using Explora-RN (CTI, USA) module. We have changed of reaction time (3, 5, 10 min) and temperature (110, 120, $130^{\circ}C$) for the optimized conditions of synthesis. Also we experimented to find the optimal concentration of precursor (5, 7, 10 mg). Results: [$^{18}F$] FHBG was purified by HPLC system and collected at around 10-12 min. Synthesis using Explora-RN module showed a $32.0{\pm}1.2%$ yield of radiochemical (decay corrected), the purity was greater than 98%. And the entire synthesis time was less than 48 min. Temperature of the highest synthesis yield was $130^{\circ}C$, reaction time was 5 minutes and concentration of precursor was 10 mg (recommended volume in manual) (n=36). In contrast to radiochemical yield of precursor 10 mg ($32{\pm}1.2%$), yield of 5 and 7 mg precursor was unstable. Conclusion: Automation of [$^{18}F$] FHBG synthesis at Explora-RN module has been completed. In addition, we were able to obtain optimized reaction time, temperature and concentration of precursor. Therefore this study would be provided more rapid synthesis and higher radiochemical yield.

• Nuclear Medicine and Molecular Imaging
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• v.40 no.4
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• pp.218-227
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• 2006

Purpose: The HSV1-tk reporter gene system is the most widely used system because of its advantage that direct monitoring is possible without the introduction of a separate reporter gene in case of HSV1-tk suicide gene therapy. In this study, we investigate the usefulness of the reporter probe (substrate), $9-(4-[^{18}F]Fluoro-3-hydroxymethylbutyl)$guanine ($[^{18}F]FHBG$) for non-invasive reporter gene imaging using PET in HSV1-tk expressing hepatoma model. Materials and Methods: Radiolabeled FHBG was prepared in 8 steps from a commercially available triester. The labeling reaction was carried out by NCA nucleophilic substitution with $K[^{18}F]/K2.2.2.$ in acetonitrile using N2-monomethoxytrityl-9-14-(tosyl)-3-monomethoxytritylmethylbutyl]guanine as a precursor, followed by deprotection with 1 N HCl. Preliminary biological properties of the probe were evaluated with MCA cells and MCA-tk cells transduced with HSV1-tk reporter gene. In vitro uptake and release-out studies of $[^{18}F]FHBG$ were performed, and was analyzed correlation between $[^{18}F]FHBG$ uptake ratio according to increasing numeric count of MCA-tk cells and degree of gene expression. MicroPET scan image was obtained with MCA and MCA-tk tumor bearing Balb/c-nude mouse model. Results: $[^{18}F]FHBG$ was purified by reverse phase semi-HPLC system and collected at around 16-18 min. Radiothemical yield was about 20-25%) (corrected for decay), radiochemical purity was >95% and specific activity was around >55.5 $GBq/{\mu}\;mol$. Specific accumulation of $[^{18}F]FHBG$ was observed in HSV1-tk gene transduced MCA-tk cells but not in MCA cells, and consecutive 1 hour release-out results showed more than 86% of uptaked $[^{18}F]FHBG$ was retained inside of cells. The uptake of $[^{18}F]FHBG$ was showed a highly significant linear correlation ($R^2=0.995$) with increasing percentage of MCA-tk numeric cell count. In microPET scan images, remarkable difference of accumulation was observed for the two type of tumors. Conclusion: $[^{18}F]FHBG$ appears to be a useful as non-invasive PET imaging substrate in HSV1-tk expressing hepatoma model.