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Patients with acute myeloid leukemia (AML) often have a poor prognosis due to drug resistance, which is regarded as a tough problem during the period of clinical therapeutics. It has been reported that autophagy, an important event in various cellular processes, plays a crucial role in mediating drug-resistance to cancer cells. Our study attempts to comparatively investigate the differences of basic and induced autophagic activity between drug-sensitive and multidrug-resistant AML cells. The level of basic autophagy in K562/ADM cells was higher than that in K562 cells, which could be characterized by more cytosolic contents-packaged autophagic vacuoles in K562/ADM cells when compared to that in K562 cells. The observation of MDC staining showed that the fluorescent intensity of autophagosomes in K562/ADM cells was stronger than that in K562 cells. The expression of Beclin1 and the ratio of LC3-II to LC3-I were distinctly higher in K562/ADM cells, however, P62 protein was relatively lower in K562/ADM cells. Furthermore, we found that nutrient depletion could induce autophagic activity of both cell lines. However, autophagic activity of K562/ADM cells was always maintained at a higher level in contrast with K562 cells. ADM (Adriamycin) was also capable of inducing autophagic activity of K562 and K562/ADM cells, but the autophagic alteration in K562 cells appeared earlier. Taken together, our findings suggest that autophagy exerts an important effect on formation and maintenance of drug-resistance in AML cells.
Acute myeloid leukemia (AML) is a highly malignant hematological neoplasm, which is characterized by high morbidity, poor prognosis and high cost of treatment, leading it to be a malignant neoplasm with one of the highest mortality rates (1). Plenty of therapeutic drugs have been used to treat patients with AML and good efficacy has been achieved (2). However, among those refractory patients, both drug resistance and poor prognosis appeared (3). Therefore, it is urgent to investigate the differences between drug-resistant and -sensitive AML cells, which may contribute to dissection of mechanisms in drug resistance.
Autophagy is an important cellular process associated with lysosome-mediated self-degradation in eukaryotic cells (4). According to different manners of substrate degradation, autophagy is mainly classified into three categories including microautophagy, chaperone-mediated autophagy (CMA) and macroautophagy (5). Macroautophagy, referred to autophagy hereinafter, exerts its function through formation of autophagosomes and subsequently being fused with lysosomes, triggering substances being degraded by acid hydrolases. Consequently, the catabolites including amino acids, fatty acids, nucleotides and so on are recycled in cytoplasm to reenter biological energy pathways, ultimately maintaining structure and metabolism of cells (6).
It has been reported that multiples of ATG (autophagy-related) proteins are recruited to tightly regulate the occurrence of autophagy, during which LC3 (microtubule- associated protein 1 light-chain 3) plays critical roles in elongation and completion of the autophagosome. In this process, the protein LC3 is cleaved at its C-terminus by Atg4B, resulting in formation of LC3-I. Subsequently, LC3-I conjugates with PE (phosphatidyl ethanolamine) and leads generation of the form of membrane-bound LC3 that called LC3-II. LC3-II is employed to the membrane of the autophagosome and specifically mediates elongation of the autophagosome. Therefore, the ratio of LC3-II to LC3-I will become larger when the quantity of autophagosomes increases (7). In addition to the shift of LC3-I to LC3-II, another crucial ATG protein P62 that interacts with ubiquitinated proteins through its UBA domain (ubiquitin-associated domain) and then delivers them to LC3-II via another domain LIR (LC3-interacting region) will be finally degraded in the autolysosome, so the level of P62 has a contrary relationship with autophagic activity (8).
Over the past few decades, basal autophagy was reported to remove misfolded proteins and damaged organelles to maintain cellular quality and health, involved in many physical processes of cells such as cellular turnover, differentiation and development (9–11). Abnormal basal autophagy can lead to various human diseases including cancer, neurodegenerative disorders, autoimmunity disease and inflammation (12–15). Autophagy can be induced in response to several stressors including: nutrient deprivation, chemotherapeutics, hypoxia, pathogen infection and endoplasmic reticulum stress (16–18). A larger body of research has reported that autophagy which is promoted by chemotherapeutic drugs in cancer cells, is related to drug-resistance of tumor cells (19–21). Autophagy is activated by doxorubicin in human colon cancer LoVo cells, and this autophagic activation reduces the sensitivity of cancer cells to doxorubicin (22). Enhanced autophagy is a survival mechanism of drug-resistant esophageal cancer cells treated with cisplatin (23). In contrast, elemene can reverse the drug resistance of the cisplatin-resistant lung adenocarcinoma cells by promoting autophagy (24). To clarify the relationship between autophagy and drug-resistance of AML cells, we tried to compare autophagic activity between K562/ADM cells and ADM-sensitive K562 cells in different environments such as the basic state, starvation and ADM treatment.
ADM was purchased from Kangbao biochemical industry company (Wuhan); HCQ (hydroxychloroquine) was bought from Tokyo Chemical Industry; MTT was obtained from Sigma; newborn bovine serum was from Rongye biotech company (Lanzhou); RPMI 1640 medium from Gibco; anti-P62, anti-LC3, anti-Beclin1 was from Cell Signaling Technology; anti-β-actin was from Biovision; HRP-linked anti-rabbit or anti-mouse IgG antibodies were from Boster biological technology company (Wuhan).
Human ADM-resistant AML cell line (K562/ADM cells) and its parental cell line (K562 cells) were both from Medical experimental center of Lanzhou University. K562/ADM and K562 cells were grown in RPMI 1640 Medium supplied with 10% inactivated newborn bovine serum and 2 mmol/L L-glutamine at 37°C in a humid atmosphere containing 5% CO2. The viability of all cultured cells was determined for up to 95% before experiments were performed.
Cells were seeded in 96-well plates at a density of 1×105 cells/mL in triplicate and were cultured with ADM at indicated concentrations and times at 37°C. MTT (5 mg/mL, 10 µL) was added to each well and cultured for another 4 h, and then 100 µL of acidifying sodium dodecyl sulfate (10%) was added to dissolve the formazan. 24 hours later, optical density (OD) was measured at 570 nm with a Powerwave X plate reader (Bio-Tek, USA). Cell proliferation inhibition rates were calculated using the following formula: cell proliferation inhibition rate = [(ODcontrol − ODexperiment)/ODcontrol] × 100% (25).
MDC has been reported to distinctively mark autophagic vesicles (26). Cells were harvested and washed twice with PBS. After resuspending in 1 mL of PBS, cells were stained with 1 µL of MDC stock-solution (50 mmol/L) of which terminal concentration reached 0.05 mmol/L for 30 min at 37°C in the dark (27). Subsequently, cells were gently washed with PBS three times and observed under an IX81 inverted microscope (Olympus, Japan).
Cells were fixed with glutaraldehyde overnight at 4°C. On the next day, after rinsing with PBS three times each for 5 min, cells were fixed for 1.5 h with osmic acid. Next, cells were rinsed with PBS again and dehydrated in the following steps: 50% alcohol for 10 min, 70% alcohol for 10 min, 90% alcohol for 10 min, 100% alcohol for 10 min, 100% acetone for 10 min twice. Subsequently, cells were embedded in an epoxy resin overnight and then solidified for 12 h at 45°C. Following slicing into sections, cells were stained with uranyl acetate and lead citrate. Finally, the ultrastructure of cells was observed under a JEM1230 transmission electron microscope (JEOL, Japan) (28).
Total RNA was isolated from cells with a Trizol kit following the manufacturer's specifications. Both the concentrations and purity of extracted RNA samples were determined by spectrophotometer. 500 ng of each RNA sample was used to synthesize cDNA with Prime Script RT Master Mix (Takara). Real-time PCR was performed with SYBR Premix Taq II (Takara) and primers. The primers used are as follows: beclin1 forward: 5′-ACCTCAGCCGAAGACTGAAG-3′, beclin1 reverse: 5′-AACAGCGTTTGTAGTTCTGAC-3′; β-actin forward: 5′-TGCTCCTCCTGAGCGCAAGTA-3′, β-actin reverse: 5′-CCACATCTGCTGGAAGGTGGA-3′. After the procedure was completed, the relative mRNA levels of genes indicated were analyzed by software Roto-Gene6.0 (29).
Cells were lysed in RIPA buffer with PMSF (PMSF:RIPA=1:100) for 30 min on ice and then centrifuged for 15 min. Protein concentration of the supernatant was measured using a BCA protein assay kit. Equal amounts of protein from cell extracts were separated by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and transferred onto a PVDF (polyvinylidene fluoride) membrane (30). The membrane was incubated with primary antibodies overnight at 4°C and then with secondary antibodies conjugated with horseradish peroxidase. Finally, the protein level was determined using a chemiluminescent approach in a dark room.
All data were statistically analyzed with the student's t-test by utilizing SPSS 17.0 software. P-value < 0.05 was considered statistically significant.
To investigate the sensitivities of K562/ADM and K562 cells to chemotherapeutic drug, we treated these two cell lines with a gradually increased concentration of ADM for different times as indicated. The MTT assay showed that ADM inhibited the growth of K562/ADM and K562 cells to various extents, and ADM-induced cytotoxicity in both cell lines was dose- and time-dependent (Figure 1A and 1B). We calculated IC50 values of ADM for K562 and K562/ADM cells, which showed that IC50 values of ADM for K562 cells for 12 h, 24 h, 48 h were 2.63 ± 0.12, 1.04 ± 0.04, 0.25 ± 0.05 µmol/L respectively and the corresponding values for K562/ADM cells were 54.19 ± 0.87, 46.46 ± 4.36, 15.44 ± 0.81 µmol/L (Figure 1C). The degree of ADM-resistance for K562/ADM cells was 20.6~62.9 fold greater when compared with that of K562 cells, suggesting K562/ADM cells have more potential for resistance to ADM than K562 cells.
ADM inhibits the proliferation of K562 cells greater than K562/ADM cells. (A) K562/ADM cells were exposed to 1.5, 3, 6, 12 and 24 µmol/L ADM for 12, 24, 48 h, and then MTT assay was performed to detect cell viability; (B) K562 cells were treated ...
To explore the relationship between autophagy and ADM resistance, we first measured the basic autophagy activities of K562/ADM and K562 cells. After observation of autophagosomes in cells under the fluorescent microscope followed by MDC staining, we found that K562 cells exhibited weak fluorescent intensity in the cytosol, whereas some bright foci could be seen in K562/ADM cells (Figure 2A). A similar result was also found using the transmission electron microscope, a gold standard for determining autophagic activity, which showed more cytosolic contents-packaged autophagic vacuoles in K562/ADM cells when compared with K562 cells (Figure 2A). To further verify the results above, we examined other autophagic indicators such as LC3 and P62/SQSTM1. As shown in Figure 2B and and2C,2C, K562/ADM cells showed a much higher ratio of LC3-II to LC3-I and relatively lower P62 levels when compared with K562 cells, indicating that K562/ADM cells had a higher level of autophagy flux. In addition, beclin1 is one of the specific genes involved in autophagy regulation. Our results displayed the level of Beclin1, mRNA expression or protein expression, was much higher in K562/ADM cells compared to K562 cells, indicating that Beclin1 might be involved in regulating basic autophagy of these two cell lines (Figure 2C and 2D).
K562/ADM cells show higher basic autophagic activity than K562 cells. (A) Autophagic morphology was observed by fluorescent microscopy followed by MDC staining (×100) (a); Autophagosomes were observed by transmission electron microscope (×8,000) ...
To compare the different autophagic variations between K562/ADM and K562 cells in nutrition depletion, we cultured the cells in serum- and amino acid-free EBSS (Earle's Balanced Salt Solution) for different times. After observing with the fluorescent microscope, we found abundant fluorescent puncta distributed in the cytosol of K562/ADM and K562 cells starved for 1h, which showed stronger fluorescent intensity relative to untreated cells (Figure 3A). A huge number of typical autophagic vesicles were also observed using the transmission electron microscope in the cytosol of starved cells (Figure 3B). Western blot results described that both the expression of Beclin1 and the ratio of LC3-II to LC3-I reached peak value in 1hour of starvation and then gradually decreased while the degradation of P62 reached peak value after 2~4 hours of starvation. However, according to LC3-II level variation, it was found that autophagic activity of K562/ADM cells always maintained a higher level in contrast with that of K562 cells (Figure 3C–3F). Additionally, the results of RT-PCR indicated beclin1 mRNA expression was enhanced in two cell lines after starvation (Figure 3G). Taken together, autophagy is induced in K562/ADM and K562 cells in a state of nutrition depletion, however, starvation-induced autophagic activity of K562/ADM cells is significantly stronger than K562 cells.
Starvation-induced autophagic activity of K562/ADM and K562 cells. (A) K562/ADM and K562 cells were cultured in EBSS for 0 h (a), 1 h (b), autophagic morphology was observed under a fluorescent microscope (×100); (B) Two cell lines were cultivated ...
To explore the different autophagic appearance in two cell lines with ADM exposure, K562/ADM and K562 cells were treated with 35 µmol/L and 0.75 µmol/L of ADM separately for 12 h or 24 h. Observing through the fluorescent microscope and transmission electron microscope, ADM enhanced the intensity of fluorescent dots and the amount of autophagic vacuoles in cytosol of both cell lines (Figure 4). The same results were also obtained in cells with exposure to HCQ, a recognized autophagy inhibitor (Figure 4). To investigate whether HCQ can inhibit ADM-induced autophagy, K562/ADM and K562 cells were pre-treated with 16 µmol/L and 4 µmol/L HCQ respectively for 3 h before exposure to ADM. We found HCQ resulted in upregulated intensity of fluorescent dots and increased autophagic vacuoles in K562/ADM and K562 cells with ADM treatment (Figure 4).
The morphological changes of autophagy in ADM-induced K562 and K562/ADM cells. K562/ADM and K562 cells were respectively treated with 35 and 0.75 µmol/L of ADM alone for 24 h, or 16 and 4 µmol/L of HCQ alone for 12 h, or were pre-treated ...
It is acknowledged that the accumulation of autophagic vacuoles is attributed either to up-regulated autophagosome generation or to down-regulated autophagosome degradation (31). As Figure 5 showed, ADM induced the rise of the LC3-II/LC3-I ratio and Beclin1 expression along with a fall of P62 level, which suggested that ADM promoted autophagy flux and the accumulation of autophagic vacuoles induced by ADM should be attributable to up-regulated autophagosome generation. However, ADM-induced autophagic alteration happened earlier in K562 cells with ADM treatment for 12 h than in K562/ADM cells exposed to ADM for 24 h. In addition, HCQ led to an increased ratio of LC3-II/LC3-I and up-regulated P62 level in the two cell lines, indicating HCQ hindered autophagy progression and the accumulation of autophagic vesicles induced by HCQ was derived from restrained degradation of autophagy. Furthermore, HCQ increased the level of LC3-II and P62, and had a diminishing effect on Beclin1 level in the case of cells exposed to ADM, implying that HCQ was capable of inhibiting ADM-induced autophagic activity by blocking the degradation of the autophagosome. The relative expression alteration of beclin1 mRNA detected by RT-PCR was consistent with that analyzed by Western blot.
ADM potentiates autophagic activity of K562/ADM and K562 cells. K562/ADM cells were exposed to ADM (35 µmol/L) alone for 12 or 24 h, or treated with HCQ (16 µmol/L) alone for 12 h, or pre-treated with HCQ for 3 h before treatment with ...
Most AML patients are prone to acquire chemotherapy resistance, which remains a troublesome obstacle for clinical therapy. Emerging evidence suggests that anti-cancer therapeutic agents can induce autophagy of cancer cells (32), which may have a vital effect on drug-resistance. Autophagy inhibition by CQ (chloroquine) significantly enhances the sensitivity of DOX-resistant human acute myelocytic leukemia HL60 cells to DOX (33). Heterogeneous nuclear ribonucleoprotein K (hnRNP K) may be associated with the development of ADM resistance in AML by augmenting autophagy (34). Inhibition of WAVE1 expression enhances the sensitivity of leukemia cells to chemotherapy by downregulating autophagy (35). All this research indicates that autophagy is a mechanism involved in chemotherapy resistance of various leukemia cells. To further elucidate this problem, it is required to analyze the differences of autophagic variation between AML chemoresistant K562/ADM cells and sensitive K562 cells.
K562/ADM cells are a multidrug-resistant cell line acquired by exposing K562 cells to step-wise increasing concentrations of ADM. It is characterized by being resistant not only to ADM, but also to mitoxantrone, homoharringtonine, rubidomycin and etoposide (36). K562/ADM cells appear to have sophisticated cross drug-resistance to all these anti-cancer drugs with quite different structures and functions. Our experiments also showed that the IC50 of K562/ADM cells was 20.6~62.9 fold greater than that of K562 cells, verifying that K562/ADM cells were highly resistant to ADM-induced proliferation inhibition and apoptosis.
Here, we found that basic autophagy in K562/ADM cells was distinctly higher than that in K562 cells, which could be indicated by more cytosolic contents-packaged autophagic vacuoles, higher Beclin1 expression level and LC3-II/LC3-I ratio, and lower P62 level in K562/ADM cells. Similar results are also obtained in a multiple myeloma cell line, Pan et al. (37) reported that expression level of LC3-II was higher in DOX-resistant RPMI8226/DOX cells than in DOX-sensitive RPMI8226/S cells. Similarly, compared with K562 cells, there is an increased PI3K/AKT/mTOR signaling and enhanced autophagic activity in imatinib-resistant human chronic myelogenous leukemia K562R cells (38).
We further investigated the different autophagic appearance between K562 and K562/ADM cells under serum depletion. A report has described that activation of autophagy is critical for cancer cellular survival under nutrient starvation (39). Another report also demonstrates that CD133-positive glioma cells exhibit higher survival under starvation conditions, which depends on induction of autophagy (40). Furthermore, autophagy induced by amino acid starvation is accelerated in the multidrug-resistant cells when compared to parental cells, resulting in enhanced cell survival capacity (41). Here, we found that starvation-induced autophagic activity of K562/ADM cells was significantly stronger relative to that of K562 cells, which may decipher why K562/ADM cells possess potent resistance to extra stressors. In conclusion, our results showed K562/ADM cells exhibited a higher basic and starvation-induced autophagy, indicating autophagy exerts a vital effect on formation and maintenance of drug-resistance in AML cells.
It is generally accepted that ADM, a canonical anthracycline chemotherapeutic drug, can induce cell death with apoptosis being involved (42). More evidence shows that ADM is also capable of inducing autophagy (43). Here we found that ADM induced autophagy in both K562/ADM and K562 cells, but the autophagic alteration in K562 cells appeared earlier, which might be attributed to its lower basic autophagic activity. In addition, HCQ suppressed autophagy flux through blocking degradation of autophagosomes. In line with this result, a recent study showed HCQ could inhibit late stage of the autophagy process by hindering lysosomal acidification (44). We also found that HCQ inhibited ADM-induced autophagic activity by restraining the degradation of the autophagosome.
Taken together, our findings suggest that autophagy exerts an important effect on formation and maintenance of drug-resistance in AML cells, however, the mechanism is still not clearly defined and is required to be investigated further and more deeply. However, our findings provide some insight into understanding the relationship between autophagy and chemoresistance in AML patients.
The present study was supported by the National Natural Science Foundation of China No. 81541025 (to HL Wei), the Fundamental Research Funds for the Central Universities No. lzujbky-2016-174 (to HL Wei) and the Natural Science Fund of Gansu No. 1208RJZA183 (to HL Wei). This work was also supported by the National Natural Science Foundation of China No. 31571196 (to L Wang), the Science and Technology Commission of Shanghai Municipality 2015 YIXUEYINGDAO project No. 15401932200 (to L Wang), the FY2008 JSPS Postdoctoral Fellowship for Foreign Researchers P08471 (to L Wang), the National Natural Science Foundation of China No. 30801502 (to L Wang), the Shanghai Pujiang Program No. 11PJ1401900 (to L Wang), Development Project of Shanghai Peak Disciplines-Integrative Medicine No. 20150407.