PSU Knowledge Bank Collection:
http://kb.psu.ac.th:80/psukb/handle/2553/1293
2024-03-28T10:41:32ZCharacterization of butyrate resistant colorectal cancer spheroid cells and its response to anticancer drugs
http://kb.psu.ac.th:80/psukb/handle/2016/19213
Title: Characterization of butyrate resistant colorectal cancer spheroid cells and its response to anticancer drugs
Authors: Kesara Nittayaboon
Abstract: Colorectal cancer (CRC) is the most common cancer worldwide, including Thailand. There are several risk factors for CRC, especially microbiome. Gut-microbiota plays a critical role in homeostasis and carcinogenesis. Butyrate, a short-chain fatty acid-producing by gut-microbiota, plays a role in intestinal homeostasis. Butyrate acts as anti-cancer agent by growth inhibition and apoptosis induction. However, microbiota study reveals that butyrate-producing bacteria were found in CRC patients more than normal and correlated to chemoresistance feature. We characterized the butyrate resistance (BR) CRC cells by treating the HCT-116 and PMF-ko14 cells line with a maximum butyrate concentration of 3.2 mM. The butyrate 50% inhibitory concentration (IC50) were increase in BR cells. The butyrate resistance mechanism was investigated with butyrate influx, and drug efflux genes expression. The increasing of influx and efflux gene expression in BR cells were found comparing to parental (PT) cells. Proteomic analysis was used to distinguish the normal and butyrate-resistant phenotype. Cell migration was used to evaluate the aggressive behavior of BR cells. The analysis reveal that HCT-BR cell show lower migration rate; however, the PMF-BR cell show higher migration rate than their PT cell. The cross-resistance to anti-cancer drugs was elucidated. We found the cross-resistance of metformin (MET) and oxaliplatin in HCT cells, and 5-fluorouracil was cross-resistance in PMF cells. Our study suggests that acquisition of resistance to butyrate induces chemoresistance in CRC cells, which may play an important role in CRC development, treatment, and metastasis. Moreover, we would like to further investigate the cytotoxicity of MET, an anti-diabetic drug with an anti-cancer activity, on PMF-BR -CRC cells in a 3D spheroid culture model. The results demonstrated that MET decreases spheroid size, viability, migration. Meanwhile, MET increases spheroid death through caspase 3/7 activity. The molecular mechanism from western blotting revealed that AMP-activated protein kinase (AMPK) and AKT serine/threonine kinase 1(Akt) were significantly upregulated in MET treatment group, whereas Acetyl-CoA-carboxylase (ACC) and mammalian target of rapamycin (mTOR) were downregulated. This situation leads to caspase activation and apoptosis. Our results confirm that MET show a potential cytotoxicity especially on the BR cells. This finding suggest that MET is an effective strategy for drug-resistant CRC cells.
Description: Doctor of Philosophy (Biomedical Sciences), 20232023-01-01T00:00:00ZEvaluation of the Synergistic Antibacterial Effects of Rifampicin-based Combination Therapies for the Management of Infections due to Carbapenem-Resistant Acinetobacter baumannii
http://kb.psu.ac.th:80/psukb/handle/2016/19158
Title: Evaluation of the Synergistic Antibacterial Effects of Rifampicin-based Combination Therapies for the Management of Infections due to Carbapenem-Resistant Acinetobacter baumannii
Authors: Nwabor, Lois Chinwe
Abstract: The increasing spread of carbapenem-resistant Acinetobacter baumanii (CRAB) is critical to public health due to the lack of treatment options and increased mortality rate. Herein, the synergistic and bactericidal effects of rifampicin in combination with conventional antibiotics were evaluated, and the molecular pathways of antibiotic resistance were predicted using bioinformatics and whole-genome sequencing. Phenotypic analysis including efflux pump detection and antibacterial activity of combination were evaluated against established biofilm cells using carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and MTT assay, respectively.
From the evaluations, about 89% of the 218 CRAB clinical isolates tested in the study showed resistance to rifampicin at zones of inhibition ≤16 mm, 9% were intermediate (17–19 mm), and 1% were susceptible (≥20 mm). The antibiotic-resistant profiles of the isolates were investigated in 31 representative clinical isolates. A total of (22/31) 71% and 94% (29/31) isolates demonstrated susceptibility to tigecycline and minocycline, respectively. The isolates showed multidrug resistance and exhibited a 100% resistance to gentamycin or tobramycin at Minimum inhibitory concentration (MIC) ≥ 1024 µg/mL, and chloramphenicol at ≥ 16µg/mL. Five isolates out of 20 were resistant to colistin at MIC = 4 µg/mL whereas 15 were intermediate at MIC=2 µg/mL. Combination therapy of rifampicin slightly improved antibiotic potency with synergism in 10/31, 7/31, 2/31, 4/31, 5/31, and 8/9 when combined with imipenem, meropenem doripenem, tigecycline, minocycline, and colistin respectively.
In addition, time-kill kinetic revealed a bactericidal effect at higher concentrations and a bacteriostatic at lower concentrations. The combination of rifampicin plus imipenem and doripenem was bactericidal against TR123 at 1/4 MIC of rifampicin and 1/4MIC of doripenem and imipenem. Rifampicin combined with tigecycline or minocycline was bactericidal at 1/4MIC rifampicin plus 1/4 MIC of the antibiotics against TR131 out of three representative isolates. Rifampicin combination with tigecycline disclosed a 2-2.5 log reduction in CFU at all the combined concentrations including at 1/4 MIC rifampicin and 1/4 MIC of tigecycline. Rifampicin plus ciprofloxacin resulted in a 99% killing against TR023 out of three isolates indicating a bactericidal activity at MIC rifampicin plus 1/2 MIC ciprofloxacin, 1/2 MIC rifampicin plus 1/2 MIC ciprofloxacin and 1/2 MIC rifampicin plus 1/4 MIC ciprofloxacin. Rifampicin plus chloramphenicol or trimethoprim-sulfamethoxazole combinations were not effective at MIC and sub- Inhibitory concentrations among all the isolates used. The combination therapy of rifampicin and fosfomycin disclosed a bacteriostatic effect against two representative isolates. Notably, rifampicin with colistin exhibited bactericidal activity in three out of four representative isolates ( PT046, TR069, and TR082) at 1/4 MIC rifampicin plus 1/4 colistin.
Antibacterial resistant mechanism assessment indicated a 4-fold reduction in the MIC of rifampicin in the presence of the efflux pump inhibitor CCCP in isolates SK056 and SK067 out of the 15 tested isolates. Biofilm viability test by MTT assay revealed a dose-dependent decrease of cell viability of established bacterial biofilm at 4 MIC rifampicin + 2 MIC carbapenems with a percentage reduction of 44–75%, compared with monotherapies at 16 MIC. The pan-genomic study of the isolates demonstrated a progressive evolution with 58% of accessory genes in the matrix. Seven of the ten sequenced isolates were of sequence type 2 (ST2), while one isolates each belongs to ST164, ST16, and ST25. Furthermore, 11 plasmids, 34 AMR genes, and 65 virulent genes were predicted to confer MDR. The blaOXA-23 blaADC-25, blaOXA-66, blaPER-7, aph(6)-Id, armA, and arr-3 were prevalent among the isolates. Sequence alignment of the bacteria genome to a reference strain revealed a deleterious mutation in the rpoB gene in 4 out of 29 isolates. Colistin-resistance-associated mutation on the PmrB and PmrC (two-component system), LPS biosynthetic protein lpxD, emrA, and emrB genes were detected among the five isolates that demonstrated resistance to colistin.
This research emphasizes the specificity of isolates to antibiotics and suggests that the rifampicin combination with colistin, tigecycline, minocycline, imipenem, meropenem, and doripenem may be a potential treatment option for the management of CRAB isolates with low rifampicin minimum inhibitory concentration. It also demonstrates that the genotypic and phenotypic characterization of antimicrobial resistance (AMR) in CRAB clinical isolates may lessen the burden of AMR surveillance.
Description: Master of Science (Biomedical Engineering), 20232023-01-01T00:00:00ZIdentification and validation of cancerous targets of kusunokinin
http://kb.psu.ac.th:80/psukb/handle/2016/19140
Title: Identification and validation of cancerous targets of kusunokinin
Authors: Tanotnon Tanawattanasuntorn
Abstract: Trans-(-)-kusunokinin hampers breast cancer growth by suppressing many target proteins, such as CFS1R and AKT. Previous results showed that it could bind other targets involved in cancer proliferation and migration. To fill in more information on the trans-(-)-kusunokinin target protein, this study used computational simulations and validated its target on breast and ovarian cancer cells. The results from molecular docking showed that AKR1B1 and MEK2 were potential targets. AKR1B1 represented the strongest binding affinity. Trans-(-)-kusunokinin indicated comparable binding affinity, interaction and orientation in the binding site to AKR1B1 inhibitors. Then, these results were indirect proof on breast (Hs578T and BT549) and ovarian (SKOV3 and A2780) cancer cells. Trans-(±)-kusunokinin had the cytotoxic effect on breast and ovarian cancer cells that were markedly stronger than well-known AKR1B1 inhibitors (zopolrestat and epalrestat). Moreover, trans-(±)-kusunokinin inhibited AKR1B1 enzyme activity with an IC50 value of 9.72 ± 0.18 uM which was stronger than trans-(-)-arctiin (13.65 ± 0.49 uM) but weaker than epalrestat (0.77 ± 0.01 uM) and zopolrestat (31.03 ± 1.40 nM). Moreover, binding between trans-(±)-kusunokinin and intracellular AKR1B1 protected the degradation of AKR1B1 at 75 C and 60 C on Hs578T and SKOV3 cells, respectively. Notably, the inhibitory effect of trans-(±)-kusunokinin on AKR1B1 led to the protection of glucose-induced cellular lipid peroxidation in a dose-dependent manner, which was stronger than epalrestat on Hs578T cells. In addition, trans-(±)-kusunokinin suppressed AKR1B1, resulting in the suppression of its signaling molecules, including PKCδ, NF-κB, AKT, Nrf2, COX2, Twist2. Trans-(±)-kusunokinin also altered epithelial mesenchymal transition (EMT) markers by increasing E-cadherin levels and decreasing N-cadherin levels on Hs578T cells. In conclusion, trans-(-)-kusunokinin exhibited a strong binding affinity with AKR1B1, thereby mitigating oxidative stress and changing EMT protein levels on aggressive breast cancer.
Description: Doctor of Philosophy (Biomedical Sciences), 20232023-01-01T00:00:00ZMass spectrometry-based serum peptidomic patterns for cervical cancer detection
http://kb.psu.ac.th:80/psukb/handle/2016/19023
Title: Mass spectrometry-based serum peptidomic patterns for cervical cancer detection
Authors: Phetploy Rungkamoltip
Abstract: Cervical cancer is the common malignancy among females worldwide,
especially in developing country. Due to high mortality and lack of sensitivity and
specificity of the screening tests (Pap smear and HPV DNA test), an alternative tool is
crucially needed. Interestingly, the peptide pattern is an alternative diagnostic tool
for discriminating healthy women from cervical cancer patients.
The serum peptide profiles from healthy subjects and cervical cancer
patients were acquired using matrix-assisted laser desorption/ionization time-of-flight
mass spectrometry (MALDI-TOF MS). A total of 222 serum samples (83 healthy
women and 139 cervical cancer patients) were divided into 2 independent datasets:
training set (30 healthy women and 75 cervical cancer patients) and validation set (53
healthy subjects and 64 cervical cancer patients). Each specimen was pooled
together according to each sample group [patients with cervical cancer
(precancerous, stage I, II and III) and healthy women]. Prior to mass spectrometry
analysis, serum peptides were prepared by ZiptipC18. Finally, the results were
analyzed using FlexAnalysis 3.0, ClinProTools 2.2 and BioTyper 2.0 software.
The serum peptide profiles in the range 1000 and 10000 Da were
analyzed and shown as the chromatogram, gel view and dimensional image of
principal component analysis (PCA). Based on peptide mass fingerprint (PMF), we
were able to discriminate and category healthy subjects and patients with stage of
cancer: precancerous, stage I, II and III. Furthermore, all PMF from pooled stage
serum were used as reference mass spectrum in the in-house database and were
used to classify samples. Compare to the clinical diagnosis, MALDI-TOF MS yields a
sensitivity (60.97%) and high specificity (93.69%). Furthermore, the peak at 1466.91 Da
was significantly differenced among the investigated groups (Wilcoxon/Kruskal-Wallis
test (pWk), p<0.001), it is, therefore, an interesting molecule for the further study.
The results of this study conclude that MALDI-TOF MS method might be used as an alternative approach for cervical cancer test with good repeatable, high accuracy, and less time-consuming.
Description: Master of Science (Biomedical Sciences), 20182018-01-01T00:00:00Z