2.1. Expression of PLK1 Is Upregulated in AML Cells and Pediatric AML Patients
As reported previously, PLK1 is highly expressed in a broad set of cancer cell lines and overexpressed in a majority of cancer patient samples compared with normal progenitor cells. However, the expression of PLK1 in AML, and specifically pediatric AML, has not been clearly defined. We demonstrate that the expression of PLK1 is very high in AML cell lines, with the highest levels observed in CCRF, NB4, and K562 cells (
Figure 1A). To examine the expression of PLK1 in pediatric AML samples, we obtained samples from 15 patients with pediatric AML and 12 control patients. High protein expression of PLK1 was observed in 73.3% (11/15) of the pediatric AML samples compared to 0% (0/12) of the normal bone marrow (NBM) control samples (
Figure 1B). Real-time PCR was also used to examine the mRNA transcript levels of PLK1 in 105 pediatric AML samples and 30 NBM/ITP (idiopathic thrombocytopenic purpura) (control samples (
Figure 1C)). PLK1 expression was significantly higher in the AML samples compared to the control samples (82.95 ± 110.28
vs. 6.36 ± 6.35;
p < 0.001). Bone marrow specimens were obtained from 105 pediatric patients with AML at the time of diagnosis, who presented at Children’s Hospital of Soochow University between 2000 and 2011. We suppose the high SD (standard deviation) values are related to the cDNA quality of samples. Examination of pediatric AML patient clinicopathology revealed that expression of PLK1 is related with FAB (French-American-Britain) and MRD (Minimal Residual Disease,
Table 1). However, there were no significant differences in other clinical features such as sex, age, initial hemoglobin level, white blood cell counts, platelet counts, or chromosomal abnormalities between individuals with high and low PLK1 expression (
Table 1). The prognostic significance of PLK1 expression was assessed in 105 Chinese pediatric AML patients with clinical follow-up records. Kaplan-Meier survival analysis revealed shorter survival times for patients with high PLK1 expression in tumors (
p = 0.002,
Table 2 and
Figure 1C). Furthermore, multivariate analysis revealed that PLK1 expression is an independent prognostic factor in pediatric AML (
p = 0.041,
Table 3). In summary, our results demonstrate that PLK1 expression is heightened in patients with pediatric AML and in human myeloid leukemia cell lines. This indicates that PLK1 may be a suitable oncogene target for pediatric AML therapy.
Figure 1.
Expression of PLK1 is upregulated in AML cells and pediatric AML patients (A) Western blot analysis showing PLK1 protein expression in nine leukemia cell lines; (B) Western blot analysis showing PLK1 protein expression in 15 pediatric AML samples and 12 NBM samples; (C) Real-time PCR analysis of the PLK1 mRNA transcript levels in 105 pediatric AML samples and 30 NBM/ITP (normal bone marrow/idiopathic thrombocytopenic purpura) control samples; and (D) Kaplan-Meier survival analysis of 105 pediatric AML patients comparing high and low PLK1 expression (p = 0.002).
Figure 1.
Expression of PLK1 is upregulated in AML cells and pediatric AML patients (A) Western blot analysis showing PLK1 protein expression in nine leukemia cell lines; (B) Western blot analysis showing PLK1 protein expression in 15 pediatric AML samples and 12 NBM samples; (C) Real-time PCR analysis of the PLK1 mRNA transcript levels in 105 pediatric AML samples and 30 NBM/ITP (normal bone marrow/idiopathic thrombocytopenic purpura) control samples; and (D) Kaplan-Meier survival analysis of 105 pediatric AML patients comparing high and low PLK1 expression (p = 0.002).
Table 1.
Association of polo-like kinase 1 (PLK1) expression with clinico-pathological characteristics in 105 pediatric acute myeloid leukemia (AML) samples.
Table 1.
Association of polo-like kinase 1 (PLK1) expression with clinico-pathological characteristics in 105 pediatric acute myeloid leukemia (AML) samples.
Clinical Variables | No. of Patients | PLK1 Expression (n) | p |
---|
Low | High |
---|
Gender | | | | |
Male | 42 | 27 | 15 | 0.373 |
Female | 63 | 35 | 28 |
Age (year) | | | | |
<6 | 60 | 35 | 25 | 0.864 |
≥6 | 45 | 27 | 18 |
Leukocyte (/μL) | | | | |
>10,000 | 61 | 37 | 24 | 0.693 |
≤10,000 | 44 | 25 | 19 |
FAB | | | | |
M1–M6 | 93 | 59 | 34 | 0.011 |
M7 | 12 | 3 | 9 |
Cytogenetics | | | | |
Favorable | 50 | 28 | 22 | 0.160 |
Intermediate | 27 | 20 | 7 |
Unfavorable | 28 | 14 | 14 |
MRD | | | | |
<0.25% | 49 | 36 | 13 | 0.005 |
≥0.25% | 56 | 26 | 30 |
Table 2.
Association of PLK1 expression with Kaplan-Meier survival in 105 pediatric AML samples.
Table 2.
Association of PLK1 expression with Kaplan-Meier survival in 105 pediatric AML samples.
Variable | No. of Patients | Over Survival | p |
---|
Median ± SE |
---|
Cytogenetics | | | |
Favorable | 50 | 46.664 ± 3.717 | <0.001 |
Intermediate | 27 | 29.220 ± 3.188 |
Unfavorable | 28 | 11.161 ± 1.827 |
FAB | | | |
M1–M6 | 93 | 36.113 ± 2.885 | <0.001 |
M7 | 12 | 8.542 ± 1.820 |
Leukocyte (/μL) | |
>10,000 | 61 | 30.220 ± 2.974 | 0.803 |
≤10,000 | 44 | 33.631 ± 4.063 |
MRD | | | |
<0.25% | 49 | 53.627 ± 3.151 | <0.001 |
≥0.25% | 56 | 18.893 ± 2.425 |
PLK1 expression | | | |
Low (<12.420) | 62 | 39.319 ± 3.539 | 0.002 |
High (≥12.420) | 43 | 24.054 ± 3.709 |
Table 3.
Cox multivariate analysis of PLK1 expression and clinico-pathological features in pediatric AML.
Table 3.
Cox multivariate analysis of PLK1 expression and clinico-pathological features in pediatric AML.
Variable | Odds Ratio | EXP (B) 95% CI | p |
---|
Cytogenetics | | | |
Favorable vs. Intermediate and Unfavorable | 6.164 | 2.477 (1.210–5.068) | 0.013 |
MRD | | | |
<0.25% vs. ≥0.25% | 14.084 | 5.176 (2.193–12.214) | 0.000 |
Leukocyte (/uL) | | | |
>10,000 vs. ≤10,000 | 0.200 | 1.138 (0.646–2.055) | 0.655 |
FAB classification | | | |
M7 vs. M1–M6 | 7.148 | 2.683 (1.301–5.533) | 0.008 |
PLK1 Expression | | | |
Low vs. High | 4.195 | 1.806 (1.026–3.179) | 0.041 |
2.2. RO3280 Inhibits the Growth of Acute Leukemia Cells
The novel PLK1 inhibitor RO3280 decreased leukemia cell viability in a dose-dependent manner (
Figure 2A,B). The RO3280 IC
50 measurement was determined in several acute leukemia cell lines: U937 186 nM, HL60 175 nM, NB4 74 nM, K562 797 nM, MV4-11 120 nM, and CCRF 162 nM. RO3280 treatment could also dramatically impact cell morphology as observed in NB4 cells (
Figure 2C).
In order to better understand the effective of RO3280, we compared it with other PLK1 inhibitors: Rigosertib (ON 01910. Na) and BI2536. The IC
50 of these PLK1 inhibitors was analyzed in both NB4 and K562 cells (
Figure 2D). In NB4 cells the following IC
50 concentrations were determined: RO3280 13.45 nM, ON 01910. Na 13.02 nM, and BI2536 87.65 nM. In K562 cells the following IC
50 concentrations were determined: RO3280 301 nM, ON 01910. Na 1606 nM, and BI2536 448 nM. To determine the effectiveness of RO3280 in primary leukemia, we determined the IC
50 in ALL and AML cells. In primary ALL the IC
50 of RO3280 is 35.49–110.76 nM (
Figure 2E,
Table 4) and in primary AML the IC
50 of RO3280 is 52.80–147.50 nM (
Figure 2E,
Table 5). These results demonstrate that the PLK1 inhibitor RO3280 effectively inhibits the proliferation of leukemia cells.
2.3. RO3280 Induced Apoptosis and Cell Cycle Disorder in Leukemia Cells
To determine if RO3280 induces apoptosis in leukemia cells, we assessed Annexin V staining, cell cycle disorder and caspase activation in leukemia cells after treatment. Cells treated with RO3280 for 24 h showed higher Annexin V staining compared with untreated cells (
Figure 3). This indicates that RO3280 induces apoptosis in leukemia cells. Cell cycle disorder was determined by a cell cycle assay, which showed that RO3280 significantly induces cell cycle disorder in nine leukemia cell lines (
Figure 4). Hoechst 33342 staining showed that 24 h RO3280 treatment induced DNA fragmentation and the formation of abnormal nuclear cells (
Figure 5A). When either HL-60 or NB4 cells were treated with RO3280, there was a significant increase in abnormal nuclear cell formation compared with control DMSO treated cells (
Figure 5B). Moreover, to clearly demonstrate that RO3280 causes apoptosis in leukemia cells, we assessed the following well-recognized markers of apoptosis: PARP, caspase-3, and caspase-9. After 24 h of treatment with RO3280 (50 or 100 nM), cleaved PARP and caspase-9 were observed in NB4 and HL-60 cells (
Figure 5C). These results are consistent with the Annexin V staining and cell cycle analysis, demonstrating that RO3280 induces apoptosis in leukemia cells. This further suggests that RO3280 may have promising antitumor therapeutic applications.
Figure 2.
RO3280 inhibits the growth of acute leukemia cells. (A) Molecular structure of RO3280; (B) Viability and IC50 analysis of RO3280 in six leukemia cells. The following are the determined RO3280 IC50s: U937 186 nM, HL60 175 nM, NB4 74 nM, K562 797 nM, MV4-11 120 nM and CCRF 162 nM; (C) Micrographs were taken of NB4 cells treated with RO3280 100 nM or DMSO (scale bar = 50 μm); (D) The IC50 of three PLK1 inhibitors, RO3280, ON 01910.Na and BI2536, was analyzed in NB4 and K562 cells. IC50 in NB4 cells: RO3280 13.45 nM, ON 01910. Na 13.02 nM, and BI2536 87.65 nM. IC50 in K562 cells: RO3280 301 nM, ON 01910. Na 1606 nM, and BI2536 is 448 nM; and (E) The IC50 of RO3280 in primary acute lymphocytic leukemia (ALL) and AML cells was also analyzed: ALL 35.49–110.76 nM and AML 52.80–147.50 nM. Cell proliferation was calculated as a percentage of the DMSO treated control wells. The IC50 values were derived after plotting proliferation values on a logarithmic curve. Experiments were performed in quadruplicate and repeated twice.
Figure 2.
RO3280 inhibits the growth of acute leukemia cells. (A) Molecular structure of RO3280; (B) Viability and IC50 analysis of RO3280 in six leukemia cells. The following are the determined RO3280 IC50s: U937 186 nM, HL60 175 nM, NB4 74 nM, K562 797 nM, MV4-11 120 nM and CCRF 162 nM; (C) Micrographs were taken of NB4 cells treated with RO3280 100 nM or DMSO (scale bar = 50 μm); (D) The IC50 of three PLK1 inhibitors, RO3280, ON 01910.Na and BI2536, was analyzed in NB4 and K562 cells. IC50 in NB4 cells: RO3280 13.45 nM, ON 01910. Na 13.02 nM, and BI2536 87.65 nM. IC50 in K562 cells: RO3280 301 nM, ON 01910. Na 1606 nM, and BI2536 is 448 nM; and (E) The IC50 of RO3280 in primary acute lymphocytic leukemia (ALL) and AML cells was also analyzed: ALL 35.49–110.76 nM and AML 52.80–147.50 nM. Cell proliferation was calculated as a percentage of the DMSO treated control wells. The IC50 values were derived after plotting proliferation values on a logarithmic curve. Experiments were performed in quadruplicate and repeated twice.
Table 4.
Pathologic features and inhibition of cell growth by RO3280 in primary culture cells of pediatric ALL.
Table 4.
Pathologic features and inhibition of cell growth by RO3280 in primary culture cells of pediatric ALL.
Number | Gender | Age (Years) | Diagnosis | ALL Typing | Chromosome Analysis | Fusion Gene | Prednisone Sensitivity | PLK1 | IC50 (nM) |
---|
1 | Female | 5 | ALL | B | 46, XY | Not detected | Sensitive | positive | 35.39 |
2 | Male | 4 | ALL | B | 46, XY | TEL/AML1+ | Sensitive | positive | 30.79 |
3 | Male | 3 | ALL | B | ALL/53–54, XY, +4, +6, +10, 12p+, +14, +17, +18, +20, +21 | Not detected | Sensitive | positive | 64.43 |
4 | Female | 4 | ALL | B | 46, XX | Not detected | Sensitive | positive | 8.27 |
5 | Female | 4 | ALL | B | ALL/53–55, XX, +X, 1q+, +4, +6, +10, +11, +15, +17, +21 | Not detected | Sensitive | positive | 36.01 |
Table 5.
Pathologic features and inhibition of cell growth by RO3280 in primary culture cells of pediatric AML.
Table 5.
Pathologic features and inhibition of cell growth by RO3280 in primary culture cells of pediatric AML.
Number | Gender | Age (Years) | Diagnosis | AML Typing | Chromosome Analysis | Fusion Gene | PLK1 | IC50 nM |
---|
1 | Female | 9 | AML | M4 | 46, XX | FLT3-ITD | positive | 88.65 |
2 | Female | 3 | AML | M4 | 46, XX, inv (16) (p13;q22) | CBF/MYH11 | positive | 47.02 |
3 | Male | 4 | AML | M5b | 46, XY, −2,+10, t (10;10) (p13;q23) | MLL/AF10 | positive | 28.57 |
4 | Male | 12 | AML | M2a | 45, X, −Y, t (8;21) (q22;q22) | AML/ETO | positive | 32.92 |
5 | Female | 1 | AML | M4 | 46, XX, inv (16) (p13;q22) | Not detected | positive | 19.20 |
Figure 3.
Annexin V analysis of apoptosis induced by RO3280 in acute leukemia cells. Annexin V staining of cells following a 24 h treatment with RO3280 at 50 or 100 nM compared with DMSO control mock treatment. All these analyses were repeated three times. ** p < 0.01.
Figure 3.
Annexin V analysis of apoptosis induced by RO3280 in acute leukemia cells. Annexin V staining of cells following a 24 h treatment with RO3280 at 50 or 100 nM compared with DMSO control mock treatment. All these analyses were repeated three times. ** p < 0.01.
Figure 4.
Cell cycle analysis of RO3280 induced cell cycle disorder in acute leukemia cells. Cell cycle analysis of nine leukemia cells treated for 24 h with RO3280 at 50 or 100 nM compared with DMSO control mock treatment. G2 phase of each group was analyzed and presented. All these analyses were repeated three times. * p < 0.05; ** p < 0.01. Left purple peak mean the G1 phase and right purple peak mean the G2 phase, red peak means the S phase; Pink, yellow and green peak mean the disorder of cell cycle.
Figure 4.
Cell cycle analysis of RO3280 induced cell cycle disorder in acute leukemia cells. Cell cycle analysis of nine leukemia cells treated for 24 h with RO3280 at 50 or 100 nM compared with DMSO control mock treatment. G2 phase of each group was analyzed and presented. All these analyses were repeated three times. * p < 0.05; ** p < 0.01. Left purple peak mean the G1 phase and right purple peak mean the G2 phase, red peak means the S phase; Pink, yellow and green peak mean the disorder of cell cycle.
Figure 5.
RO3280 induced DNA fragmentation and cleavage of apoptotic markers in acute leukemia cells. (A) Micrographs following Hoechst 33342 staining of cells treated with RO3280 (50 and 100 nM) for 24 h. This demonstrates the induction of DNA fragmentation and abnormal nuclear cell formation; (B) The abnormal nuclear cells were quantified and increased significantly with RO3280 treatment compared with mock treatment in both HL-60 and NB4 cells. ** p < 0.01; and (C) Western blot analysis of PARP, caspase-3, and caspase-9. After 24 h treatment with 50 or 100 nM RO3280, cleaved PARP and caspase-9 were detected in lysates from NB4 and HL-60 cells.
Figure 5.
RO3280 induced DNA fragmentation and cleavage of apoptotic markers in acute leukemia cells. (A) Micrographs following Hoechst 33342 staining of cells treated with RO3280 (50 and 100 nM) for 24 h. This demonstrates the induction of DNA fragmentation and abnormal nuclear cell formation; (B) The abnormal nuclear cells were quantified and increased significantly with RO3280 treatment compared with mock treatment in both HL-60 and NB4 cells. ** p < 0.01; and (C) Western blot analysis of PARP, caspase-3, and caspase-9. After 24 h treatment with 50 or 100 nM RO3280, cleaved PARP and caspase-9 were detected in lysates from NB4 and HL-60 cells.
2.4. Real-Time PCR Array Identifies Genes Implicated in the Effect of RO3280 Treatment
In order to identify apoptosis and/or programmed cell death molecules implicated in the effects of RO3280, we used the SABioscience Human Apoptosis PCR Array PAHS-3012. With this real-time PCR array we analyzed and clustered the expression of 370 genes associated with apoptosis in DMSO or RO3280 treated cells (
Figure 6A). The genes most significantly upregulated or downregulated are shown in
Figure 6B,C, respectively. Examination of the array data revealed that 32 genes were significantly upregulated and 16 genes were significantly downregulated in RO3280 treatment group compared with DMSO control group (
Table 6 and
Table 7, respectively). The upregulated genes included protein kinase C zeta, receptor-interacting serine-threonine kinase 3, harakiri BCL2 interacting protein, DCC netrin 1 receptor, and cyclin-dependent kinase inhibitor 1A. The downregulated genes included interleukin 1α, nucleotide-binding oligomerization domain containing 2, caspase-1, apoptosis-related cysteine peptidase, serpin peptidase inhibitor clade B, B-cell CLL/lymphoma 2, and Bruton agammaglobulinemia tyrosine kinase. The RO3280-dependent upregulation of DCC and CDKN1A and downregulation of BTK and SOCS2 was verified by western blot analysis (
Figure 7).
Figure 6.
Real-time PCR array identifies genes implicated in the effect of RO3280 treatment. (A) Gene expression clustering of 370 key apoptosis genes in 50 nM RO3280-treated NB4 cells compared to DMSO-treated cells; (B) Relative expression of the most upregulated genes in RO3280-treated NB4 cells compared to DMSO-treated cells; and (C) Relative expression of the most downregulated genes in RO3280-treated NB4 cells compared to DMSO-treated cells.
Figure 6.
Real-time PCR array identifies genes implicated in the effect of RO3280 treatment. (A) Gene expression clustering of 370 key apoptosis genes in 50 nM RO3280-treated NB4 cells compared to DMSO-treated cells; (B) Relative expression of the most upregulated genes in RO3280-treated NB4 cells compared to DMSO-treated cells; and (C) Relative expression of the most downregulated genes in RO3280-treated NB4 cells compared to DMSO-treated cells.
Figure 7.
Western blot verification of real-time PCR array results. Western blot analysis of cells following a 24 h treatment with RO3280 at 50 or 100 nM compared with DMSO control mock treatment. The upregulation of DCC and CDKN1A and down regulation of BTK and SOCS2 in RO3280 treated cells. Protein lysates from treated cells were tested for expression levels by Western blot analysis.
Figure 7.
Western blot verification of real-time PCR array results. Western blot analysis of cells following a 24 h treatment with RO3280 at 50 or 100 nM compared with DMSO control mock treatment. The upregulation of DCC and CDKN1A and down regulation of BTK and SOCS2 in RO3280 treated cells. Protein lysates from treated cells were tested for expression levels by Western blot analysis.
Table 6.
Genes upregulated in NB4 cells treated with RO3280 compared with the DMSO control group.
Table 6.
Genes upregulated in NB4 cells treated with RO3280 compared with the DMSO control group.
Gene | Description | DMSO | RO3280 | Fold Change | p Value |
---|
GDNF | glial cell derived neurotrophic factor | 0.0029 | 9.4172 | 3262.8632 | 0.0162 |
PRKCZ | protein kinase C, zeta | 0.0042 | 12.7679 | 3065.9848 | 0.0457 |
RIPK3 | receptor-interacting serine-threonine kinase 3 | 0.0025 | 4.5249 | 1802.2433 | 0.0126 |
HRK | harakiri, BCL2 interacting protein | 0.1471 | 150.6503 | 1023.9242 | 0.0246 |
GRM4 | glutamate receptor, metabotropic 4 | 0.3625 | 335.0571 | 924.1889 | 0.0225 |
TDGF1 | teratocarcinoma-derived growth factor 1 | 0.0451 | 27.3203 | 605.1673 | 0.0218 |
DCC | DCC netrin 1 receptor | 3.4002 | 425.7855 | 125.2247 | 0.0225 |
BDNF | brain-derived neurotrophic factor | 44.1739 | 1023.2023 | 23.1630 | 0.0007 |
EEF1A2 | eukaryotic translation elongation factor 1 α 2 | 633,377.6354 | 12,905,456.2968 | 20.3756 | 0.0083 |
NUPR1 | nuclear protein, transcriptional regulator, 1 | 11.4562 | 232.1343 | 20.2629 | 0.0001 |
CIDEC | cell death-inducing DFFA-like effector c | 33.4482 | 667.6981 | 19.9621 | 0.0187 |
CDKN1A | cyclin-dependent kinase inhibitor 1A | 2113.7362 | 36,484.2997 | 17.2606 | 0.0113 |
BNIP-2 | BCL2/adenovirus E1B 19 kDa interacting protein 2 | 18.2415 | 306.7483 | 16.8160 | 0.0145 |
BIRC5 | baculoviral IAP repeat containing 5 | 60,340.7297 | 981,719.5648 | 16.2696 | 0.0164 |
TIMP3 | TIMP metallopeptidase inhibitor 3 | 15.1670 | 201.9090 | 13.3124 | 0.0031 |
CASP14 | caspase 14, apoptosis-related cysteine peptidase | 32.1570 | 410.5166 | 12.7660 | 0.0226 |
CARD10 | caspase recruitment domain family, member 10 | 28,245.9365 | 329,683.7209 | 11.6719 | 0.0292 |
ALOX15B | arachidonate 15-lipoxygenase, type B | 68.4589 | 796.1735 | 11.6299 | 0.0488 |
APOE | apolipoprotein E | 411.8114 | 4632.2654 | 11.2485 | 0.0119 |
BCL3 | B-cell CLL/lymphoma 3 | 950.2097 | 7763.8336 | 8.1707 | 0.0013 |
BNIP3L | BCL2/adenovirus E1B 19 kDa interacting protein 3-like | 16,818.9831 | 130,854.4287 | 7.7802 | 0.0001 |
DAPK1 | death-associated protein kinase 1 | 40.6424 | 287.7947 | 7.0811 | 0.0050 |
AIFM2 | apoptosis-inducing factor, mitochondrion-associated, 2 | 1759.5718 | 12,454.0471 | 7.0779 | 0.0156 |
BCL6 | B-cell CLL/lymphoma 6 | 385.7559 | 2574.1201 | 6.6729 | 0.0061 |
AIFM3 | apoptosis-inducing factor, mitochondrion-associated, 3 | 764.9698 | 5057.6217 | 6.6115 | 0.0500 |
ZNF443 | zinc finger protein 443 | 2001.5333 | 12,744.6420 | 6.3674 | 0.0009 |
DAPK3 | death-associated protein kinase 3 | 5607.9528 | 30,206.6219 | 5.3864 | 0.0020 |
BCL2L11 | BCL2-like 11 (apoptosis facilitator) | 7094.6609 | 34,581.3650 | 4.8743 | 0.0000 |
BIRC-2 | baculoviral IAP repeat containing 2 | 8366.5954 | 35,580.9121 | 4.2527 | 0.0007 |
BAG3 | BCL2-associated athanogene 3 | 10,487.7923 | 34,964.8259 | 3.3339 | 0.0002 |
Bcl-10 | B-cell CLL/lymphoma 10 | 13,170.6013 | 36,171.7865 | 2.7464 | 0.0000 |
BFAR | bifunctional apoptosis regulator | 14,398.6434 | 31,271.8562 | 2.1719 | 0.0243 |
Table 7.
Genes downregulated in NB4 cells treated with RO3280 compared with the DMSO control group.
Table 7.
Genes downregulated in NB4 cells treated with RO3280 compared with the DMSO control group.
Gene | Description | DMSO | RO3280 | Fold Change | p Value |
---|
IL1A | interleukin 1α | 39.8703 | 0.0000 | 0.0000 | 0.0316 |
NOD2 | nucleotide-binding oligomerization domain containing 2 | 135.5059 | 0.0000 | 0.0000 | 0.0000 |
CASP1 | caspase 1, apoptosis-related cysteine peptidase | 161.0668 | 0.0011 | 0.0000 | 0.0001 |
NME5 | NME/NM23 family member 5 | 26.4650 | 0.0006 | 0.0000 | 0.0000 |
SERPINB2 | serpin peptidase inhibitor, clade B | 168.2487 | 0.2504 | 0.0015 | 0.001 |
SOCS2 | suppressor of cytokine signaling 2 | 150.3483 | 2.5012 | 0.0166 | 0.0006 |
MPO | myeloperoxidase | 1,652,217.4204 | 42,599.1776 | 0.0258 | 0.0000 |
TNFSF15 | tumor necrosis factor superfamily, member 15 | 15.2009 | 1.2803 | 0.0842 | 0.0360 |
Bcl2A1 | BCL2-related protein A1 | 619.9195 | 152.6633 | 0.2463 | 0.0019 |
Bcl-2 | B-cell CLL/lymphoma 2 | 31,688.9516 | 8210.5905 | 0.2591 | 0.0026 |
BTK | Bruton’s agammaglobulinemia tyrosine kinase | 37,873.9816 | 9837.2022 | 0.2597 | 0.0001 |
IL31RA | interleukin 31 receptor A | 823.6783 | 230.4415 | 0.2798 | 0.0009 |
PROK2 | prokineticin 2 | 13,280.3733 | 4801.4889 | 0.3615 | 0.0004 |
AZU1 | azurocidin 1 | 29,361.9342 | 10,755.3769 | 0.3663 | 0.0003 |
LTBR | lymphotoxin β receptor | 6702.9536 | 2753.6837 | 0.4108 | 0.0004 |
HSPA9 | heat shock 70 kDa protein 9 | 318,280.1754 | 141,258.3887 | 0.4438 | 0.0000 |
Serine/threonine-protein kinase PLK1, also known as polo-like kinase 1 (PLK-1) or serine/threonine-protein kinase 13 (STPK13). PLK1 is an early trigger for G2/M transition. PLK1 supports the functional maturation of the centrosome in late G
2/early prophase and establishment of the bipolar spindle. PLK1 is an interesting molecule both as a biomarker and as a target for highly specific cancer therapy for several reasons. Firstly, it is over-expressed in many cancers and can serve as a biomarker. PLK1 is highly expressed in a broad range of tumors. Secondly, the expression of PLK1 in untransformed cells is not nearly as high, which makes it a discriminating target for the development of cancer-specific small molecule drugs. Medical experts are now paying more attention to the role of PLK1 in acute myeloid leukemia [
35]. Here we show that PLK1 is highly expressed in all AML cell lines tested and in 73.3% (11/15) of the pediatric AML samples compared to 0% (0/12) of the NBM control sample. Real-time PCR analysis of 105 pediatric AML samples demonstrated significantly upregulated PLK1 expression in the AML samples compared to the control samples. Sometimes the results of the RT-PCR were not very accurate when the cDNA quality of samples was not very high. Thus, it is best to utilize Western blot analysis in future studies. Kaplan-Meier survival analysis of 105 pediatric AML patients revealed shorter survival time correlated with high PLK1 expression in tumors. Furthermore, multivariate analysis revealed that PLK1 expression is an independent prognostic factor in pediatric AML. In summary, our results are indicate for the first time that PLK1 may be a good oncogene target in samples from pediatric AML patients and human myeloid leukemia cell lines.
Several PLK1 inhibitors have been discovered and show significant anticancer effects. ATP-competitive inhibitor BI 6727 [
36,
37] is currently in clinical trials for cancer treatment. PLK1 inhibition with BI2536 leads to apoptotic effects in SCCHN cell lines [
38]. BI2536 is a potent PLK1 inhibitor with IC
50 of 0.83 nM. BI2536 also shows 4- and 11-fold greater selectivity against PLK2 and PLK3, and its effects in leukemia have been reported by others [
32]. TAK-960 is currently undergoing Phase I evaluation in adult patients with advanced solid malignancies [
39]. ON 01910. Na inhibits mitotic progression and induces apoptosis in most cancer cell lines [
40], is currently in a phase I study for treating adult patients with advanced solid malignancies [
41]. Rigosertib (ON-01910) is a PLK1 inhibitor with IC
50 of 9 nM. As presented here, in leukemia cells, RO3280 is more effective than Rigosertib (ON-01910). Wovkulich and colleagues recently developed RO3280, a novel PLK1 inhibitor [
30]. RO3280 is a potent, highly selective inhibitor of PLK1 with IC
50 of 3 nM. Until now, we know that PLK1 is a target of RO3280 and RO3280 has nearly no effect on PLK2 and PLK3. However, the effectiveness of RO3280 in leukemia treatment remains an open question. In this report we demonstrate that RO3280 inhibits leukemia cell proliferation in a dose-dependent manner and causes apoptosis. This suggests that RO3280 may be a promising antitumor treatment for leukemia. Indeed the potential of PLK1 inhibitors in treatment is promising, and combinations of PLK1 inhibitors with other anticancer drugs might offer a larger range of opportunities for cancer therapy [
42].
Real-time PCR Array System is ideal tool for analyzing the expression of a focused panel of genes [
43]. In this study, we used real-time PCR arrays to analyze the effect of RO3280 treatment on apoptotic gene regulation. Compared to an untreated control group, 32 genes related to apoptosis were significantly upregulated and 16 genes were significantly downregulated after RO3280 treatment. This confirmed previous reports that CDKN1A, CASP1, and BCL2 are regulated by PLK1 inhibition. PLK1 is a checkpoint protein whose role spans all of mitosis and includes DNA repair. PLK1 has been reported as a negative regulator of CDKN1A (p21) [
44]. When cancer cells are treated with PLK1 inhibitors, p21 is increased in the cytoplasm. By contrast, deficiency of p21 renders tumor cells more susceptible to Polo-like kinase 1 inhibition [
45]. A rrecent study showed that PLK1 inhibition causes post-mitotic DNA damage and senescence in a range of human tumor cell lines [
46]. Small-molecule PLK1 inhibitors and PLK1 genetic knock-down resulted in the induction of DNA double-strand breaks and senescence in a range of human tumor cell lines. Molecular function of PLK1 is very complicated and in metastatic alveolar rhabdomyosarcoma (aRMS), PLK1 interacted with and phosphorylated PAX3-FOXO1 leading to protein stabilization. PLK1 inhibition led to elevated ubiquitination and rapid proteasomal degradation of this PAX3-FOXO1 chimeric oncoprotein [
47]. Our study also identified some novel genes such as RIPK3, HRK, DCC, and BTK regulated by PLK1 inhibition. While each of these genes has a function related to apoptosis, many are additionally involved in separate cellular processes. RIPK3 is a component of the tumor necrosis factor (TNF) receptor-I signaling complex and a key regulator of TNF-induced necrosis [
48]. The DCC gene encodes a netrin 1 receptor that partially localizes to lipid rafts and induces apoptosis in the absence of ligand. The receptor functions as a tumor suppressor and is frequently mutated or down regulated in colorectal [
49] and ovarian cancers [
50]. HRK promotes apoptosis in mammalian cells by interacting with the apoptotic inhibitors BCL-2 and BCL-X
L via its BH3 domain [
51]. Recent research indicates that PLK1 is an important regulator of PTEN during cell cycle progression [
52]. Our research now indicates that PLK1 may also regulate RIPK3, DCC, and HRK expression. Until now, the regulation mechanism of PLK1 is still unclear. Thus, our results may provide new clues into the molecular mechanism of apoptosis induced by RO3280 in leukemia cells.