• Molecules and Cells
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• 제39권3호
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• pp.217-228
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• 2016

To generate a biobetter that has improved therapeutic activity, we constructed scFv libraries via random mutagenesis of several residues of CDR-H3 and -L3 of hu4D5. The scFv clones were isolated from the phage display libraries by stringent panning, and their antiproliferative activity against HER2-positive cancer cells was evaluated as a primary selection criterion. Consequently, we selected AH06 as a biobetter antibody that had a 7.2-fold increase in anti-proliferative activity ($IC_{50}$: 0.81 nM) against the gastric cancer cell line NCI-N87 and a 7.4-fold increase in binding affinity ($K_D$: 60 pM) to HER2 compared to hu4D5. The binding energy calculation and molecular modeling suggest that the substitution of residues of CDR-H3 to W98, F100c, A101 and L102 could stabilize binding of the antibody to HER2 and there could be direct hydrophobic interactions between the aromatic ring of W98 and the aliphatic group of I613 within HER2 domain IV as well as the heavy and light chain hydrophobic interactions by residues F100c, A101 and L102 of CDR-H3. Therefore, we speculate that two such interactions were exerted by the residues W98 and F100c. A101 and L102 may have a synergistic effect on the increase in the binding affinity to HER2. AH06 specifically binds to domain IV of HER2, and it decreased the phosphorylation level of HER2 and AKT. Above all, it highly increased the overall level of p27 compared to hu4D5 in the gastric cancer cell line NCIN82, suggesting that AH06 could potentially be a more efficient therapeutic agent than hu4D5.

• Biomolecules & Therapeutics
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• 제27권5호
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• pp.492-501
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• 2019

Nitrogen-containing heterocycles such as quinoline, quinazolinones and indole are scaffolds of natural products and have broad biological effects. During the last years those structures have been intensively synthesized and modified to yield new synthetic molecules that can specifically inhibit the activity of dysregulated protein kinases in cancer cells. Herein, a series of newly synthesized isoquinolinamine (FX-1 to 8) and isoindoloquinazolinone (FX-9, FX-42, FX-43) compounds were evaluated in regards to their anti-leukemic potential on human B- and T- acute lymphoblastic leukemia (ALL) cells. Several biological effects were observed. B-ALL cells (SEM, RS4;11) were more sensitive against isoquinolinamine compounds than T-ALL cells (Jurkat, CEM). In SEM cells, metabolic activity decreased with $10{\mu}M$ up to 26.7% (FX-3), 25.2% (FX-7) and 14.5% (FX-8). The 3-(p-Tolyl) isoquinolin-1-amine FX-9 was the most effective agent against B- and T-ALL cells with IC50 values ranging from 0.54 to $1.94{\mu}M$. None of the tested compounds displayed hemolysis on erythrocytes or cytotoxicity against healthy leukocytes. Anti-proliferative effect of FX-9 was associated with changes in cell morphology and apoptosis induction. Further, influence of FX-9 on PI3K/AKT, MAPK and JAK/STAT signaling was detected but was heterogeneous. Functional inhibition testing of 58 kinases revealed no specific inhibitory activity among cancer-related kinases. In conclusion, FX-9 displays significant antileukemic activity in B- and T-ALL cells and should be further evaluated in regards to the mechanisms of action. Further compounds of the current series might serve as templates for the design of new compounds and as basic structures for modification approaches.

• 한국미생물·생명공학회지
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• 제29권3호
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• pp.181-185
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• 2001

유방암 세포주에서는 우수한 항암활성을 가진 것으로 알려진 indole-3-carbinol을 HepG2세포주에 시간과 농도별로 처리한 결과 cell growth inhibition을 확인하였으며, $IC_{50}$ 값은 48시간배양에서 $446\mu$M 72시간 배양에서 444$\mu$M로 나타났다. $400\mu$M의 I3C을 투여하고, 24, 48, 72시간에 HePG2 세포주의 cell cycle pattern을 분석한 결과, G1 phase에서 P21의증가와 함께 Cdk 6와 cyclin D의 확연한 감소와 Pb protein의 hypo-phosphorylation을 확인하였다. 반면 G2 phase에서는 I3C의 직접적인 억제로 인해 24시간 후부터 Cdc2와 cyclin B1가 급격히 감소하는것을 확인하였다. Flow cytomery 분석결과 I3C 처리 24시간 뒤 G2 arrest (25%)가 발생하였으며, 72시간이 지난후 G1 arrest (53%)가 발생하였다. 이러한 I3C의 간암세포주인 HePG2 cell의 cell cycle arrest가 apoptosis를 유발하는지를 알고자 caspase 3 Bcl2 Bax protein의 발현양상을 확인한 결과 아무런 변화가 보이지 않았다. 즉 I3C은 간암세포주인 HepG2 cell에서 apoptosis를 유도하지 못한다는것을 확인하였따. 결론적으로 I3C은 HepG2 세포주에서 G1와 G2 phase에서 cell cycle arrest는 발생시키나, 특이적으로 apoptosis 와는 연관되지 않는다는 사실을 확인하였다.