• Biomolecules & Therapeutics
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• v.32 no.4
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• pp.481-491
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• 2024

Paxlovid is the first approved oral treatment for coronavirus disease 2019 and includes nirmatrelvir, a protease inhibitor targeting the main protease (M^pro) of SARS-CoV-2, as one of the key components. While some specific mutations emerged in M^pro were revealed to significantly reduce viral susceptibility to nirmatrelvir in vitro, there is no report regarding resistance to nirmatrelvir in patients and animal models for SARS-CoV-2 infection yet. We recently developed xenograft tumors derived from Calu-3 cells in immunodeficient mice and demonstrated extended replication of SARS-CoV-2 in the tumors. In this study, we investigated the effect of nirmatrelvir administration on SARS-CoV-2 replication. Treatment with nirmatrelvir after virus infection significantly reduced the replication of the parental SARS-CoV-2 and SARS-CoV-2 Omicron at 5 days post-infection (dpi). However, the virus titers were completely recovered at the time points of 15 and 30 dpi. The virus genomes in the tumors at 30 dpi were analyzed to investigate whether nirmatrelvir-resistant mutant viruses had emerged during the extended replication of SARS-CoV-2. Various mutations in several genes including ORF1ab, ORF3a, ORF7a, ORF7b, ORF8, and N occurred in the SARS-CoV-2 genome; however, no mutations were induced in the M^pro sequence by a single round of nirmatrelvir treatment, and none were observed even after two rounds of treatment. The parental SARS-CoV-2 and its sublineage isolates showed similar IC₅₀ values of nirmatrelvir in Vero E6 cells. Therefore, it is probable that inducing viral resistance to nirmatrelvir in vivo is challenging differently from in vitro passage.

• Korean Journal of Radiology
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• v.21 no.6
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• pp.726-735
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• 2020

Objective: Recent innovations in biology are boosting gene and cell therapy, but monitoring the response to these treatments is difficult. The purpose of this study was to find an MRI-reporter gene that can be used to monitor gene or cell therapy and that can be delivered without a viral vector, as viral vector delivery methods can result in long-term complications. Materials and Methods: CMV promoter-human organic anion transporting polypeptide 1B3 (CMV-hOATP1B3) cDNA or CMV-blank DNA (control) was transfected into HEK293 cells using Lipofectamine. OATP1B3 expression was confirmed by western blotting and confocal microscopy. In vitro cell phantoms were made using transfected HEK293 cells cultured in various concentrations of gadoxetic acid for 24 hours, and images of the phantoms were made with a 9.4T micro-MRI. In vivo xenograft tumors were made by implanting HEK293 cells transfected with CMV-hOATP1B3 (n = 4) or CMV-blank (n = 4) in 8-week-old male nude mice, and MRI was performed before and after intravenous injection of gadoxetic acid (1.2 µL/g). Results: Western blot and confocal microscopy after immunofluorescence staining revealed that only CMV-hOATP1B3-transfected HEK293 cells produced abundant OATP1B3, which localized at the cell membrane. OATP1B3 expression levels remained high through the 25th subculture cycle, but decreased substantially by the 50th subculture cycle. MRI of cell phantoms showed that only the CMV-hOATP1B3-transfected cells produced a significant contrast enhancement effect. In vivo MRI of xenograft tumors revealed that only CMV-hOATP1B3-transfected HEK293 tumors demonstrated a T1 contrast effect, which lasted for at least 5 hours. Conclusion: The human endogenous OATP1B3 gene can be non-virally delivered into cells to induce transient OATP1B3 expression, leading to gadoxetic acid-mediated enhancement on MRI. These results indicate that hOATP1B3 can serve as an MRI-reporter gene while minimizing the risk of long-term complications.

• Journal of Veterinary Science
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• v.21 no.2
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• pp.26.1-26.14
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• 2020

Pancreatic ductal adenocarcinoma is a lethal cancer type that is associated with multiple gene mutations in somatic cells. Genetically engineered mouse is hardly applicable for developing a pancreatic cancer model, and the xenograft model poses a limitation in the reflection of early stage pancreatic cancer. Thus, in vivo somatic cell gene engineering with clustered regularly interspaced short palindromic repeats is drawing increasing attention for generating an animal model of pancreatic cancer. In this study, we selected Kras, Trp53, Ink4a, Smad4, and Brca2 as target genes, and applied Campylobacter jejuni Cas9 (CjCas9) and Streptococcus pyogens Cas9 (SpCas9) for developing pancreatic cancer using adeno associated virus (AAV) transduction. After confirming multifocal and diffuse transduction of AAV2, we generated SpCas9 overexpression mice, which exhibited high double-strand DNA breakage (DSB) in target genes and pancreatic intraepithelial neoplasia (PanIN) lesions with two AAV transductions; however, wild-type (WT) mice with three AAV transductions did not develop PanIN. Furthermore, small-sized Cjcas9 was applied to WT mice with two AAV system, which, in addition, developed high extensive DSB and PanIN lesions. Histological changes and expression of cancer markers such as Ki67, cytokeratin, Mucin5a, alpha smooth muscle actin in duct and islet cells were observed. In addition, the study revealed several findings such as 1) multiple DSB potential of AAV-CjCas9, 2) peri-ductal lymphocyte infiltration, 3) multi-focal cancer marker expression, and 4) requirement of > 12 months for initiation of PanIN in AAV mediated targeting. In this study, we present a useful tool for in vivo cancer modeling that would be applicable for other disease models as well.

• Journal of Digestive Cancer Reports
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• v.5 no.2
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• pp.97-104
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• 2017

Background: Cachexia is a multi-factorial syndrome presenting with chronic illness, decreases in body weight, and loss of adipose tissue and skeletal muscle, mostly in patients with advanced cancer and chronic wasting disease. Even after years of intensive researches, there remains no convincing therapy to prevent cancer cachexia. Methods: In this in vivo study, we have established C26 adenocarcinoma-induced cancer cachexia model in mice to explore the underlying core changes in cytokine, signal transduction, and muscle wasting. The ultimate aim of establishing animal model is to find optimal therapeutics to mitigate cancer cachexia. Results: We have administered C26 adenocarcinoma cells onto BALB/c mice and observed 4 weeks to assess the progression of cancer cachexia. Significant loss of weight accompanied with loss of appetite was noted. As C26 adenocarcinoma xenograft progressed, mortality was started from 3 weeks, accompanied with significant sarcopenia and decreased mice movement. Surges in TNF-α and IL-6 were noted with the commencement of cancer cachexia. Conclusion: Using C26 adenocarcinoma cancer cachexia model, we can screen the optimal therapeutics to mitigate cancer cachexia, in which agents to modulate IL-6, TNF-α, and NF-κB were essential.

• Journal of Digestive Cancer Research
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• v.5 no.2
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• pp.97-104
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• 2017

Background: Cachexia is a multi-factorial syndrome presenting with chronic illness, decreases in body weight, and loss of adipose tissue and skeletal muscle, mostly in patients with advanced cancer and chronic wasting disease. Even after years of intensive researches, there remains no convincing therapy to prevent cancer cachexia. Methods: In this in vivo study, we have established C26 adenocarcinoma-induced cancer cachexia model in mice to explore the underlying core changes in cytokine, signal transduction, and muscle wasting. The ultimate aim of establishing animal model is to find optimal therapeutics to mitigate cancer cachexia. Results: We have administered C26 adenocarcinoma cells onto BALB/c mice and observed 4 weeks to assess the progression of cancer cachexia. Significant loss of weight accompanied with loss of appetite was noted. As C26 adenocarcinoma xenograft progressed, mortality was started from 3 weeks, accompanied with significant sarcopenia and decreased mice movement. Surges in TNF-α and IL-6 were noted with the commencement of cancer cachexia. Conclusion: Using C26 adenocarcinoma cancer cachexia model, we can screen the optimal therapeutics to mitigate cancer cachexia, in which agents to modulate IL-6, TNF-α, and NF-κB were essential.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.9
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• pp.3901-3906
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• 2014

Aim: The purpose of this study was to investigate anti-tumor effects and safety of DH332, a new ${\beta}$-carboline alkaloids derivatives in vitro and in vivo. Materials and Methods: The effects of DH332 on human (RAMOS RA.1) and mouse (J558) B lymphoma cell lines were detected using a CCK-8 kit (Cell Counting Kit-8), and apoptosis was detected by flow cytometry with PI/annexinV staining. Western blotting was used to detected caspase-3 and caspase-8. Neurotoxic and anti-tumor effects were evaluated in animal experiments. Results: DH332 exerts a lower neurotoxicity compared with harmine. It also possesses strong antitumor effects against two B cell lymphoma cell lines with low $IC_{50s}$. Moreover, DH332 could inhibit the proliferation and induce the apoptosis of RAMOS RA.1 and J558 cell lines in a dose-dependent manner. Our results suggest that DH332 triggers apoptosis by mainly activating the caspase signaling pathway. In vivo studies of tumor-bearing BALB/c mice showed that DH332 significantly inhibited growth of J558 xenograft tumors. Conclusions: DH332 exerts effective antitumor activity in vitro and in vivo, and has the potential to be a promising drug candidate for lymphoma therapy.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.4
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• pp.2361-2366
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• 2013

Diverse studies have shown that miR-155 is overexpressed in different tumor types. However, the precise molecular mechanism of the ectopic expression of miR-155 in breast cancer is still poorly understood. To further explore the role of miR-155 in breast tumorigenesis, we here assessed the influence of miR-155 antisense oligonucleotide (miR-155 ASO) on MDA-MB-157 cell viability and apoptosis in vitro. Furthermore, the effects of inhibitory effects of miR-155 on the growth of xenograft tumors in vivo were determined with performance of immunohistochemistry to detect expression of caspase-3, a pivotal apoptosis regulatory factor, in xenografts. Transfection efficiency detected by laser confocal microscope was higher than 80%. The level of miR-155 expression was significantly decreased (P<0.05) in the cells transfected with miR-155 ASO, compared with that in cells transfected with a negative control. After being transfected with miR-155 ASO, the viability of MDA-MB-157 cells was reduced greatly (P<0.05) and the number of apoptotic cells was increased significantly. Additionally, miR-155 ASO inhibited the growth of transplanted tumor in vivo and significantly increased the expression of caspase-3. Taken together, our study revealed that miR-155 ASO can induce cell apoptosis and inhibit cell proliferation in vitro. Moreover, miR-155 ASO could significantly repress tumor growth in vivo, presumably by inducing apoptosis via caspase-3 up-regulation. These findings provide experimental evidence for using miR-155 as a therapeutic target of breast carcinoma.

• The Journal of Internal Korean Medicine
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• v.25 no.1
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• pp.92-105
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• 2004

In order to evaluate the anti-tumor and synergic effect of BoJungIkKeeHapDaeChilKi-Tang on doxorubicin, the inhibitory concentration(IC), IC50 and IC90 of single use of doxorubicin and BoJungIkKeeHapDaeChilKi-Tang with their concomitant treatment against MKN-45(Human stomach carcinoma) was observed using MTT(Microculture Tetrazolium test) assay. In addition, their anti-tumor effects were also observed in the xenograft nude mice models agianst MKN-45 cell lines. BoJungIkKeeHapDaeChilKi-Tang has only mimic direct anti-tumor effect against to MKN-45 cell lines but they were decreased general depressed signs induced by implantation of tumor cell lines and increased the total WBC and lymphocyte numbers. So, it is considered or expected that BoJungIkKeeHapDaeChilKi-Tang extracts were reduced by the critical toxicity of doxorubicin and shows favorable synergic effect with doxorubicin and BoJungIkKeeHapDaeChilKi-Tang extracts.

• Herbal Formula Science
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• v.12 no.2
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• pp.119-137
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• 2004

In order to evaluate the anti-tumor and synergic effect of Soonkiwhajungtang with doxorubicin, the inhibitory concentration(IC), $IC_{50}\;and\;IC_{90}$ of single use of doxorubicin and Soonkiwhajungtang with their concomitant treatment against Colon-26(Murine Rectum Carcinoma) was observed using MTT(Microculture Tetrazolium test) assay. In addition, their anti-tumor effects were also observed in the xenograft nude mice models agianst to Colon-26 cell lines. Soonkiwhajungtang has only mimic direct anti-tumor effect against to Colon-26 cell lines but they were decreased general depressed signs induced by implantation of tumor cell lines and increased the total WBC and lymphocyte numbers. So, it is considered or expected that Soonkiwhajungtang extracts were reduced the critical toxicity of doxorubicin and shows favorable synergic effect with doxorubidn and Soonkiwhajungtang extracts.

• The Journal of Internal Korean Medicine
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• v.25 no.2
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• pp.183-194
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• 2004

In order to evaluate the anti-tumor and synergic effect of Soonkiwhajungtang with doxorubicin, the inhibitory concentration(IC), IC50 and IC90 of single use of doxorubicin and Soonkiwhajungtang with their concomitant treatment against Colon-26(Murine Rectum Carcinoma) was observed using MTT(Microculture Tetrazolium test) assay. In addition, their anti-tumor effects were also observed in the xenograft nude mice models against 3LL cell lines. Soonkiwhajungtang may only mimic direct anti-tumor effects against 3LL cell lines, but signs of worsening induced by implantation of tumor cell lines generally decreased, while the total WBC and lymphocyte numbers increased. Therefore, experimentation suggests that Soonkiwhajungtang extracts reduced the critical toxicity of doxorubicin, and that Soonkiwhajungtang extracts have favorable synergic effects when combined with doxorubicin.