Human Chronic Myelogenous Leukemia Cell Research Articles

Association of poly(rC)-binding protein-2 with sideroflexin-3 through TOM20 as an iron entry pathway to mitochondria

Iron is essential for all the lives and mitochondria integrate iron into heme and Fe-S clusters for diverse use as cofactors. Here, we screened mitochondrial proteins in KU812 human chronic myelogenous leukemia cells by glutathione S-transferase pulldown assay with PCBP2 to identify mitochondrial receptors for PCBP2, a major cytosolic Fe(II) chaperone. LC–MS analyses identified TOM20, sideroflexin-3 (SFXN3), SFXN1 and TOM70 in the affinity-score sequence. Stimulated emission depletion microscopy and proteinase-K digestion of mitochondria in HeLa cells revealed that TOM20 is located in the outer membrane of mitochondria whereas SFXN3 is located in the inner membrane. Although direct association was not observed between PCBP2 and SFXN3 with co-immunoprecipitation, proximity ligation assay demonstrated proximal localization of PCBP2 with TOM20 and there was a direct binding between TOM20 and SFXN3. Single knockdown either of PCBP2 and SFXN3 in K562 leukemia cells significantly decreased mitochondrial catalytic Fe(II) and mitochondrial maximal respiration. SFXN3 but not MFRN1 knockout (KO) in mouse embryonic fibroblasts decreased FBXL5 and heme oxygenase-1 (HO-1) but increased transferrin uptake and induced ferritin, indicating that mitochondrial iron entry through SFXN3 is distinct. MFRN1 KO revealed more intense mitochondrial Fe(II) deficiency than SFXN3 KO. Insufficient mitochondrial heme synthesis was evident under iron overload both with SFXN3 and MFRN KO, which was partially reversed by HO-1 inhibitor. Conversely, SFXN3 overexpression caused cytosolic iron deficiency with mitochondrial excess Fe(II), which further sensitized HeLa cells to RSL3-induced ferroptosis. In conclusion, we discovered a novel pathway of iron entry into mitochondria from cytosol through PCBP2-TOM20-SFXN3 axis.

Biochips to measure ultraviolet light absorption by leukemia cells

During the treatment of leukemia, side effects create a great burden for patients. Therefore, new therapies to replace conventional chemotherapy and hematopoietic stem cell transplantation are highly desirable. In a previous study, I found that UV irradiation at 365 nm induced apoptosis in 98% of leukemia (Jurkat T) cells. To determine the most suitable wavelength for such treatments, it is necessary to measure the UV light absorption of cancer cells. In this study, a biochip was fabricated and evaluated for this purpose. Three types of leukemic cells (human acute myelogenous leukemia cells, human acute lymphoblastic leukemia cells, and human chronic myelogenous leukemia cells) were injected into the biochip, and light absorption was measured, revealing that individual leukemic cells have different light absorption characteristics. This variation is important in order to develop an implant care device to treat leukemia more effectively.

Inhibition of LATS kinases reduces tumorigenicity and increases the sensitivity of human chronic myelogenous leukemia cells to imatinib

Chronic myelogenous leukemia (CML) is a clonal hematologic malignancy of the myeloid lineage caused by the oncogenic BCR/ABL fusion protein that promotes CML cell proliferation and protects them against drug-induced apoptosis. In this study, we determine LATS1 and LATS2 expression in CML cells derived from patients who are resistant to imatinib (IM) treatment. Significant upregulation of LATS1 and LATS2 was found in these CML patients compared to healthy donors. To further explore whether the expression of LATS1/2 contributes to the IM-resistant phenotype, IM-resistant CML cell lines generated by culturing CML-derived erythroblastic K562 cells in increasing concentrations of IM were used as in vitro models. Up-regulation of LATS1 and LATS2 was observed in IM-resistant K562 cells. Reduction of LATS using either Lats-IN-1 (TRULI), a specific LATS inhibitor, or shRNA targeting LATS1/2 significantly reduced clonogenicity, increased apoptosis and induced differentiation of K562 cells to late-stage erythroid cells. Furthermore, depletion of LATS1 and LATS2 also increased the sensitivity of K562 cells to IM. Taken together, our results suggest that LATS could be one of the key factors contributing to the rapid proliferation, reduced apoptosis, and IM resistance of CML cells. Targeting LATS could be a promising treatment to enhance the therapeutic effect of a conventional BCR/ABL tyrosine kinase inhibitor such as IM.

L‐lysine Janus Dendrimer Conjugate with Naproxen and Mefenamic Acid and its Anticancer Activity

AbstractDendritic architectures have potential use as excellent carriers for drug delivery due to their multifunctionality, biopermeability, and biodegradability properties. Furthermore, their conjugation with non‐steroidal anti‐inflammatory drugs (NSAIDs) results in a synergetic effect improving their anticancer activity. Here we report the synthesis and cytotoxic activity of novel Janus dendrimer conjugates with naproxen and mefenamic acid moieties at the periphery. Diethanolamine dendrons were convergently synthesized through a protection‐deprotection sequence with naproxen as a terminal group and finally coupled to triethylene glycol as a linker. On the other hand, mefenamic acid was covalently joined to the L‐lysine. The dendron and dendrimer‐conjugates with naproxen and mefenamic acid showed high inhibition and selectivity against PC‐3 (human prostatic adenocarcinoma), and K‐562 (human chronic myelogenous leukemia) cell lines, but low cytotoxicity against normal cell lines. To explain the molecular mechanisms involved in the anticancer activity of dendrimer conjugates, our research will be continued.

Tyrosine Kinase Inhibitor Profiling Using Multiple Forskolin-Responsive Reporter Cells.

We have developed a highly sensitive promoter trap vector system using transposons to generate reporter cells with high efficiency. Using an EGFP/luciferase reporter cell clone responsive to forskolin, which is thought to activate adenylate cyclase, isolated from human chronic myelogenous leukemia cell line K562, we found several compounds unexpectedly caused reporter responses. These included tyrosine kinase inhibitors such as dasatinib and cerdulatinib, which were seemingly unrelated to the forskolin-reactive pathway. To investigate whether any other clones of forskolin-responsive cells would show the same response, nine additional forskolin-responsive clones, each with a unique integration site, were generated and quantitatively evaluated by luciferase assay. The results showed that each clone represented different response patterns to the reactive compounds. Also, it became clear that each of the reactive compounds could be profiled as a unique pattern by the 10 reporter clones. When other TKIs, mainly bcr-abl inhibitors, were evaluated using a more focused set of five reporter clones, they also showed unique profiling. Among them, dasatinib and bosutinib, and imatinib and bafetinib showed homologous profiling. The tyrosine kinase inhibitors mentioned above are approved as anticancer agents, and the system could be used for similarity evaluation, efficacy prediction, etc., in the development of new anticancer agents.

Design, synthesis of Janus dendrons conjugated with ketoprofen and prednisone for human chronic myelogenous therapy

Herein we present the synthesis of ketoprofen dendrons of first and second generation, with oxobutanoic acid chain lengths, a β-d-galactose derivative, an alkyne terminal group and the other dendrons with prednisone-triethylene glycol with an azide terminal group. Finally, the Janus dendrons were obtained by a coupling azide-alkyne “click” reaction. The cytotoxicity in vitro of the dendrons was evaluated against six different cancer cell lines and only showed activity against human chronic myelogenous leukemia (K-562) cells, the African green monkey kidney (COS-7) cell line was used as reference. The IC50 of the dendrons of first and second generation against K-562 was from 51.29 ± 0.1 μM, 47.17 ± 0.7 μM and 37.15 ± 0.6 μM respectively. However, the Janus dendron of first and second generation showed IC50 (13.43 ± 0.5 μM and 7.02 ± 0.2 μM) respectively. The most interesting result was obtained with the Janus dendrimer of second generation, which was found to be the best inhibitor of this series against the K-562 cell line.

Methyl-Thiol-Bridged Oxadiazole and Triazole Heterocycles as Inhibitors of NF-κB in Chronic Myelogenous Leukemia Cells.

Nuclear factor kappa beta (NF-κB) is a transcriptional factor that plays a crucial role in regulating cancer cell proliferation. Therefore, the inhibition of NF-κB activity by small molecules may be beneficial in cancer therapy. In this report, methyl-thiol-bridged oxadiazole and triazole heterocycles were synthesized via click chemistry and it was observed that the lead structure, 2-(((1-(3,4-dichlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)thio)-5-(4-methoxybenzyl)-1,3,4-oxadiazole (4c), reduced the viability of MCF-7 cells with an IC50 value of 7.4 µM. Compound 4c also caused concentration-dependent loss of cell viability in chronic myelogenous leukemia (CML) cells. Furthermore, compound 4c inhibited the activation of NF-κB in human CML cells as observed by nuclear translocation and DNA binding assays. Functionally, compound 4c produced PARP cleavage and also suppressed expression of Bcl-2/xl, MMP-9, COX-2, survivin, as well as VEGF, resulting in apoptosis of CML cells. Moreover, ChIP assay showed that compound 4c decreased the binding of COX-2 to the p65 gene promoter. Detailed in silico analysis also indicated that compound 4c targeted NF-κB in CML cells. In conclusion, a novel structure bearing both triazole and oxadiazole moieties has been identified that can target NF-κB in CML cells and may constitute a potential novel drug candidate.

Anticancer Activity of 3,5-Bis(dodecyloxy)Benzoate-PAMAM Conjugates with Indomethacin or Mefenamic Acid.

The synthesis of conjugates with nonsteroidal anti-inflammatory drugs could improve their activity with less toxicity and these compounds could be used for the treatment of cancer. The aim of the present investigation was the synthesis of 3,5-bis(dodecyloxy)benzoate - PAMAM conjugates with indomethacin and mefenamic acid to examine their anticancer activity. The anticancer activity was studied of the conjugates against six human cancer cells U- 251, PC-3, K-562, HCT-15, MCF-7, SKLU-1, and the COS-7 (as a control) cell lines. The conjugates with indomethacin and mefenamic acid were characterized by 1H, 13C NMR one- and twodimension spectroscopy. All the conjugates synthetized with indomethacin or mefenamic acid showed anticancer activity against all the human cancer cell lines. The first generation of indomethacin conjugates showed better activity against the PC-3 (human prostatic adenocarcinoma) cell line than the second generation. But the second generation with indomethacin showed better activity against PC-3 than the first generation. The second-generation conjugate with mefenamic acid had strong selectivity to PC-3 cells with an IC50 value of 10.23 ± 1.2 μM in vitro. In the paper, we report the synthesis and spectroscopic analyses of new indomethacin or mefenamic acid conjugates. The overall results showed that the conjugate of the second generation with mefenamic acid could be a potential nanocarrier for human prostatic adenocarcinoma cancer treatment, our research will be continued.

Anti-Cancer Effects of Oxygen-Atom-Modified Derivatives of Wasabi Components on Human Leukemia Cells.

1-Isothiocyanato-6-(methylsulfinyl)-hexanate (6-MITC) is a natural compound found in Wasabia japonica. The synthetic derivatives 1-Isothiocyanato-6-(methylsulfenyl)-hexane (I7447) and 1-Isothiocyanato-6-(methylsulfonyl)-hexane (I7557) were obtained from 6-MITC by deleting and adding an oxygen atom to the sulfone group, respectively. We previously demonstrated that extensive mitotic arrest, spindle multipolarity, and cytoplasmic vacuole accumulation were induced by 6-MITC and inhibited the viability of human chronic myelogenous leukemia K562 cells. In this study, we examined the anti-cancer effects of 6-MITC derivatives on human chronic myelogenous leukemia (CML) cells. Autophagy was identified as the formation of autophagosomes with double-layered membranes using transmission electron microscopy. Cell cycle and differentiation were analyzed using flow cytometry. Apoptosis was detected by annexin V staining. After treatment with I7447 and I7557, the G2/M phase of cell cycle arrest was revealed. Cell death can be induced by a distinct mechanism (the simultaneous occurrence of autophagy and aberrant mitosis). The expression levels of acridine orange were significantly affected by lysosomal inhibitors. The natural wasabi component, 6-MITC, and its synthetic derivatives have similar effects on human chronic myelogenous leukemia cells and may be developed as novel therapeutic agents against leukemia.

Abstract 3832: Apoptotic and cell cycle effects of triterpenes isolated from Phoradendron wattii on a chronic myelogenous leukemia cell line

Abstract Introduction: Medicinal plants have been used in traditional medicine practices since ancient times. Mayan traditional medicine uses herbs as treatment to illness that can be related to cancer disease such as Phoradendron wattii. As part of the search for novel cancer treatment two triterpene compounds were isolated from P. wattii: 3-epi-betulinic acid and lupenone with preliminary activity on leukemias. We investigated the cancer treatment abilities of 3-epi-betulinic acid and lupenone on human chronic myelogenous leukemia (K562) cell line and monocytes isolated from peripheral blood using flow cytometry methods. Results: Lupenone showed low effect on both cells with an IC50 of 177.60 ± 5.07 µg/mL and a selectivity index (SI) of 0.10 times more on K-562 cell line than on monocytes. Nevertheless, 3-epi-betulinic acid showed a moderate anti-CML activity with an IC50 of 38.74 ± 5.07 µg/mL, having a SI of 4.10 on K-562 cells when compared to monocytes. These results indicate that 3-epi-betulinic acid was 41 times more selective for K-562 cell line than lupenone and it also was 7.45 more selective than etoposide as positive control. Additionally, 3-epi-betulinic acid increased cell population in the G0/G1 phase of the cell cycle and apoptotic cells, and it inhibited cell proliferation in a dose-dependent manner. Furthermore, the relative expression of genes associated with apoptosis Bax and caspase 3 increased compared to control. Conclusion: Combined, these results suggest that 3-epi-betulinic acid plays an important regulatory role of cell cycle and apoptosis. Finally, the 3-epi-betulinic acid investigated in this work is potentially a cheap, safe, and effective alternative to chemotherapy in cancer treatment. Citation Format: Rosa E. Moo-Puc, Shirley A. Calderón Sauri, Jairo R. Villanueva-Toledo, Sergio R. Peraza-Sánchez, Antonieta Chávez-González. Apoptotic and cell cycle effects of triterpenes isolated from Phoradendron wattii on a chronic myelogenous leukemia cell line. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3832.

NI-BODIPY-GO Nanocomposites for Targeted PDT.

Three multifunctional targeted NI-BODIPYs (10-12) and GO-(10-12) nanocarriers were fabricated. NI-BODIPYs are designed to facilitate non-covalent interaction with graphene oxide (GO) and target toward cancer cells for specific recognition with glucose moieties while efficiently producing singlet oxygen. We probed detailed characterization, fundamental photophysical/photochemical properties, and interactions with GO of such triplet photosensitizers and nanocarriers. The effect of the formation of nanohybrids with GO on singlet oxygen formation as well as on the efficacies of the molecules in terms of in vitro killing of cancer cells was evaluated with K562 human chronic myelogenous leukemia cells. Amazingly, it was observed that GO exhibited favorable interactions with the NI-BODIPY dyads and promoted the formation of singlet oxygen, while not showing any dark toxicity.

Antileukemia and Anticolorectal Cancer Activity of Janus Dendrimer Conjugates with Naproxen and Ibuprofen

AbstractThe synthesis of a non‐steroidal anti‐inflammatory dendritic drug carrier, naproxen and ibuprofen‐based poly‐ester Janus dendrimer was carried out using a conventional azide‐alkyne click‐chemistry reaction. The corresponding dendron linkers contain naproxen and ibuprofen drug moieties on the surface and a stable [1,2,3]‐triazole as focal nucleus. The Janus dendrimer conjugate with four naproxen in one face and four ibuprofen on the other face was very active against human chronic myelogenous leukemia (K‐562) and human colorectal adenocarcinoma (HCT‐15) cells compared with cisplatin. This design provided an effective pathway for the synthesis of multiple drug carriers with two or more different end drugs in the structure.

A novel tumor-targeting strain of Xenorhabdus stockiae exhibits potent biological activities.

Xenorhabdus are symbionts of soil entomopathogenic nematodes of the genus Steinernema presenting two distinct forms in their life cycle, and can produce a broad range of bioactive compounds. In this study, a novel Xenorhabdus stockiae strain HN_xs01 was isolated from a soil sample via an entrapment method using Galleria melonella nematodes. The supernatants of strain HN_xs01 exhibited antimicrobial properties against Gram-negative and Gram-positive bacteria, and insecticidal properties against Helicoverpa armigera larvae, and antitumor properties as well. Moreover, three linear rhabdopeptides (1, 2 and 3) were identified from strain HN_xs01 using nuclear magnetic resonance analysis, which exhibited significant cytotoxic activity against the human epithelial carcinoma cell line A431 and the human chronic myelogenous leukemia cell line K562. Some bacteria have been reported to colonize the tumor region, and we determined that HN_xs01 could grow in tumor xenografts in this study. HN_xs01 invaded and replicated in B16 melanoma cells grafted into C57BL/6 mice, resulting in tumor inhibition. Moreover, strain HN_xs01 not only merely aggregated in the tumor environment, but also prevented pulmonary metastasis. It caused fragmentation of vessels and cell apoptosis, which contributed to its antitumor effect. In conclusion, X. stockiae HN_xs01 is a novel tumor-targeting strain with potential applications in medicinal and agricultural fields.

Chemical characterization of non-psychoactive Cannabis sativa L. extracts, in vitro antiproliferative activity and induction of apoptosis in chronic myelogenous leukaemia cancer cells.

In this study, extracts from non‐psychoactive Cannabis sativa L. varieties were characterized by means of ultra high‐performance liquid chromatography coupled with high‐resolution mass spectrometry (UHPLC‐HRMS) and their antiproliferative activity was assessed in vitro. The human chronic myelogenous leukaemia cell line K562 was chosen to investigate the mechanism of cell death. The effect on the cell cycle and cell death was analysed by flow cytometry. Proteins related to apoptosis were studied by western blotting. Mechanical properties of cells were assessed using the Micropipette Aspiration Technique (MAT). The results indicated that the cannabidiol (CBD)‐rich extract inhibited cell proliferation of K562 cell line in a dose‐dependent manner and induced apoptosis via caspase 3 and 7 activation. A significant decrease in the mitochondrial membrane potential was detected, together with the release of cytochrome c into the cytosol. The main apoptotic markers were not involved in the mechanism of cell death. The extract was also able to modify the mechanical properties of cells. Thus, this hemp extract and its pure component CBD deserve further investigation for a possible application against myeloproliferative diseases, also in association with other anticancer drugs.

Designed hybrid anticancer nuclear-localized peptide inhibits aggressive cancer cell proliferation.

Cell proliferation is a crucial step that might promote cancer if deregulated. Therefore, this vital segment is critically controlled by a complicated cell-cycle process in normal cells that is regulated by some regulatory proteins. It has been observed that p16 protein, playing a crucial role in cell-cycle progression/regulation, remains inactivated in different cancer cells. This inactivity of p16 protein leads to the enhancement of cancer cell proliferation by allowing uncontrolled cancer cell division. Hence, the activity of p16 protein needs to be restored using new viral vectors, small molecules as well as peptides to control/suppress this type of abnormal cell proliferation. In this work, we have taken an interesting approach to increase the efficiency and bio-availability of p16 peptide (functional part of p16 protein) to be an aggressive anti-leukemia therapeutic agent by conjugating a nuclear-localized signal (NLS) sequence and a short peptide (AVPI) with it. Moreover, this newly designed NLS attached hybrid peptide greatly affects XIAP expressing but p16 lower expressing human chronic myelogenous leukemia (CML) cell proliferation by targeting both nuclear (CDK4/cyclin D) and cellular factors (XIAP) and promoting the caspase-3 dependent apoptosis pathway.

Inula graveolens induces selective cytotoxicity in glioblastoma and chronic leukemia cells.

Crude oil extracts, components of extracts, and ethanolic extracts of Inula graveolens possess various pharmacological activities on various cancer cells including antioxidative and antiproliferative effects. Aqueous extract of this species has not been investigated on the liquid malignancies and solid tumors with a high incidence of treatment refractoriness and poor survival outcomes such as glioblastoma and leukemia. Hence, the present study aimed to evaluate the cytotoxic efficiency of I. graveolens aqueous extracts on human glioblastoma multiforme and chronic myelogenous leukemia cell lines in comparison to non-cancerous primary rat cerebral cortex and human peripheral blood mononuclear cells. The cells were treated with the extracts of I. graveolens (125-1000 μg/mL) for 48 h, the cellular viability was identified using 3'-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, and lactate dehydrogenase release was measured to determine the cytotoxic potential. Total oxidant status and apurinic/apyrimidinic endodeoxyribonuclease 1 assays were used to determine the oxidative status of cells and DNA damage, respectively. I. graveolens showed selective cytotoxicity toward human glioblastoma multiforme and chronic myelogenous leukemia cell lines and exhibited a higher antiproliferative effect against cancer cells in comparison to non-cancerous cells. Moreover, it significantly reduced the apurinic/apyrimidinic endodeoxyribonuclease 1 levels on both cancer cell lines as compared with their control cells without changing the levels of an oxidative stress marker. The extracts of I. graveolens have anti-cancer potential on human glioblastoma multiforme and chronic myelogenous leukemia cell lines without causing oxidative stress.

Transcriptome Analysis of Chronic Myelogenous Leukemia Cell Line with Imatinib Resistance

To investigate the regulation of chronic myelogenous leukemia （CML） imatinib resistant genes, in order to improve the therapeutic effect of CML imatinib resistant patients. The human CML cell line K562 and imatinib-resistant K562 cells (K562/G01) were collected, and transcriptome of the cells were achieved by RNA-seq. The sequencing data were analyzed by using standard procedures. Compared with K562 cells, 464 genes were significantly changed in K562/G01 cells, including 163 up-regulated and 301 down-regulated genes. The GO function annotation analysis and KEGG pathway analysis results showed that the differentially expressed genes were mainly involved in biological processes such as oxidative phosphorylation, localization to protein organelle, ribonucleoprotein complex biogenesis and so on. Gene Set Enrichment Analysis (GSEA) plots showed that 5 gene-sets were up-regulated in K562/G01 significantly, including the pathway of TGF-beta, mTOR and CML. CML imatinib resistance is associated with oxidative phosphorylation, during which the pathway of TGF-beta and mTOR are significantly up-regulated.

Kaempferol sensitizes tumor necrosis factor-related apoptosis-inducing ligand-resistance chronic myelogenous leukemia cells to apoptosis.

The tumor necrosis factor (TNF)-related apoptosis-inducing ligand, TRAIL, an apoptosis-inducing cytokine, has attracted much attention in the treatment of cancer for its selective toxicity to malignant rather than normal cells. However, the apoptosis-inducing ability of TRAIL is weaker than expected primarily due to cancer cell resistance. As one of the dietary flavonoids, kaempferol, has been shown to be antiproliferative and might have a protective effect against TRAIL resistance, particularly for hematologic malignancies. Here, we studied the potential of kaempferol to enhance the TRAIL-induced cytotoxicity and apoptosis in human chronic myelogenous leukemia (CML) cell line K-562, as well as the expression of specific genes with impact on TRAIL signal regulation. Analysis of flowcytometry data showed that treatment with kaempferol did enhance sensitivity of CML cells to pro-apoptotic effects of anti-TRAIL antibody. Although the gene expression levels were heterogeneous, cFLIP, cIAP1 and cIAP2 expression were generally downregulated where co-treatment of kaempferol and TRAIL was employed and these effects appeared to be dose-dependent. We further demonstrated that the expression of death receptors 4 and 5 tended to increase subsequent to the combination treatment. Consequently, it is reasonable to conclude that sensitization of chronic leukemia cells to TRAIL by kaempferol in vitro should be considered as a way of focusing clinical attention on leukemia therapy.

JKST6, a novel multikinase modulator of the BCR-ABL1/STAT5 signaling pathway that potentiates direct BCR-ABL1 inhibition and overcomes imatinib resistance in chronic myelogenous leukemia

Chronic myelogenous leukemia (CML) is a hematological malignancy that highly depends on the BCR-ABL1/STAT5 signaling pathway for cell survival. First-line treatments for CML consist of tyrosine kinase inhibitors that efficiently target BCR-ABL1 activity. However, drug resistance and intolerance are still therapeutic limitations in Ph+ cells. Therefore, the development of new anti-CML drugs that exhibit alternative mechanisms to overcome these limitations is a desirable goal. In this work, the antitumoral activity of JKST6, a naphthoquinone-pyrone hybrid, was assessed in imatinib-sensitive and imatinib-resistant human CML cells. Live-cell imaging analysis revealed JKST6 potent antiproliferative activity in 2D and 3D CML cultures. JKST6 provoked cell increase in the subG1 phase along with a reduction in the G0/G1 phase and altered the expression of key proteins involved in the control of mitosis and DNA damage. Rapid increases in Annexin V staining and activation/cleavage of caspases 8, 9 and 3 were observed after JKST6 treatment in CML cells. Of interest, JKST6 inhibited BCR-ABL1/STAT5 signaling through oncokinase downregulation that was preceded by rapid polyubiquitination. In addition, JKST6 caused a transient increase in JNK and AKT phosphorylation, whereas the phosphorylation of P38-MAPK and Src was reduced. Combinatory treatment unveiled synergistic effects between imatinib and JKST6. Notably, JKST6 maintained its antitumor efficacy in BCR-ABL1-T315I-positive cells and CML cells that overexpress BCR-ABL and even restored imatinib efficacy after a short exposure time. These findings, together with the observed low toxicity of JKST6, reveal a novel multikinase modulator that might overcome the limitations of BCR-ABL1 inhibitors in CML therapy.

Unveiling the Molecular Dynamics in a Living Cell to the Subcellular Organelle Level Using Second-Harmonic Generation Spectroscopy and Microscopy.

Second-harmonic generation (SHG) microscopy has been proved to be a powerful method for investigating the structures of biomaterials. SHG spectra were also generally used to probe the adsorption and cross-membrane transport of molecules on lipid bilayers in situ and in real time. In this work, we applied SHG and two-photon fluorescence (TPF) spectra to investigate the dynamics of an amphiphilic ion with an SHG and TPF chromophore, D289 (4-(4-diethylaminostyry)-1-methyl-pyridinium iodide), on the surface of human chronic myelogenous leukemia (K562) cells and the subcellular structures inside the cells. The adsorption and cross-membrane transport of D289 into the cells and then into the organelles such as mitochondria were revealed. SHG images were also recorded and used to demonstrate their capability of probing molecular dynamics in organelles in K562 cells. This work demonstrated the first SHG investigation of the cross-membrane transport dynamics on the surface of subcellular organelles. It may also shed light on the differentiation of different types of subcellular structures in cells.