Choline kinase α (CHKα)

MN58b had an IC50 of 3.14 μM against parental Suit2 007 cells and 0.77 μM against gemcitabine-conjugated cells [1]. At 1 μM and 5 μM growth, MN58b (1-5 μM; 72 hours; SK-PC-1, Suit2 008, IMIM-PC2, and RWP-1 cells) significantly affected colony formation in all cell lines[1]. SK-PC-1, Suit2 008, IMIM; MN58b ((1-10 μM; 24-48 hours).

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The antiproliferative activity of the CHKαI MN58b is associated with CHKα expression levels [1]
MN58b selectively inhibits CHKα; accordingly, we observed a decrease in the synthesis of phosphocholine from choline in IMIM-PC-2 cells (Fig. 4A). We analyzed the effects of MN58b on the growth of four PDAC cell lines (SK-PC-1, Suit2 008, IMIM-PC-2, and RWP-1). MN58b had a marked effect on colony formation at 1 μmol/L, and growth was completely abolished at 5 μmol/L in all the cell lines tested (Fig. 4B). In a panel of 12 PDAC cell lines, the IC50 of MN58b ranged from 0.23 to 3.2 μmol/L. We found a direct relationship between CHKα protein expression and MN58b sensitivity (R2 = 0.88; Fig. 4B and Supplementary Table S3). CHKα knockdown in Suit2 028 and Suit2 007 cells was associated with an increase in the IC50 (Supplementary Table S4).

To determine the mechanism of action of MN58b, we treated PDAC cells with increasing concentrations of MN58b (1–10 μmol/L) for 24 or 48 hours and analyzed apoptosis through Annexin V. There was a direct correlation between CHKα expression and the percentage of Annexin V–positive cells at 48 hours (Fig. 4C). The induction of apoptosis was confirmed through the analysis of cleaved caspase-9 by western blotting; a dose–response relationship was observed (data not shown). Therefore, MN58b induces apoptosis and this response correlates with CHKα expression. These results suggest that CHKα could be a predictive marker of response to MN58b.

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Combination effects of MN58b and chemotherapeutic agents [1]
Both primary and acquired resistances contribute to the limited efficacy of gemcitabine in the treatment of PDAC. We used parental and gemcitabine-resistant Suit2 007 cells to assess the relationship between resistance and MN58b sensitivity. The IC50 of MN58b for parental and resistant cells was 3.14 μmol/L and 0.77 μmol/L, respectively, supporting the notion that MN58b could be a therapeutic alternative in gemcitabine-resistant tumors.

To test the synergism of MN58b with other chemotherapeutic agents active in PDAC therapy (5), we treated PDAC cells (SK-PC-1, Suit2 028, and RWP-1) expressing variable levels of CHKα with gemcitabine, oxaliplatin, or 5-FU plus MN58b at concentrations lower than the IC50. The synergism was measured as CI. In Suit2 028 cells, none of the combinations tested showed increased effects. In the other two cell lines, MN58b showed an additive effect in combination with gemcitabine and 5-FU, and synergism in combination with oxaliplatin (SK-PC-1, CI = 0.23; RWP-1, CI = 0.39; Fig. 4D and Supplementary Fig. S4). These findings support the use of MN58b in combination with other chemotherapeutic drugs.

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Resistance to MN58b is mediated by the upregulation of the ABCB transporters 1 and 4 [1]
To assess the mechanisms involved in the acquisition of resistance to CHKI, we generated an MN58b-resistant line from parental IMIM-PC-2 cells by continuous culture with increasing drug concentrations. After 9 months of treatment, IMIM-PC-2-R cells were established; their IC50 was 156 μmol/L, approximately 30-fold higher than that of parental cells. Colony-forming capacity of IMIM-PC-2-R cells was not affected by treatment with 10 μmol/L MN58b (Fig. 5A). IMIM-PC-2-R displayed a lower baseline proliferation rate than parental cells (Fig. 5A) as well as reduced choline uptake (approximately 50%; Fig. 5B). However, CHKα enzymatic activity was similar in resistant and parental cells (Fig. 5C).

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Cellular effects of MN58b treatment. [2]
MDA-MB-231 and HT29 cells were treated with MN58b at pharmacologically active concentrations corresponding to 5 × IC50 obtained by the 96 hours of sulforhodamine B assay (6 and 2.5 μmol/L, respectively) for 4, 13, 19, 30, and 48 hours (MDA-MB-231) and 48 hours (HT29). After 48 hours of incubation, the number of MN58b treated cells per flask as a % of control showed a statistically significant reduction to 78 ± 10% (P < 0.04) in MDA-MB-231 (Fig. 1A) and 48 ± 5% (P < 0.01) in HT29, consistent with decreased proliferation. In contrast, following treatment with the inactive analogue ACG20b, cell number was similar to controls (95 ± 8%, P = 0.5) and (95 ± 7%, P = 0.4) in MDA-MB-231 and HT29 respectively. To further characterize the cellular effects of MN58b, cell cycle distribution following MN58b treatment was determined by flow cytometry on attached cells. No statistically significant effect on the cell cycle distribution was seen in MDA-MB-231 cells following treatment with MN58b for up to 30 hours. However, at 48 hours, the percentage of cells in G1 phase showed a statistically significant increase from 43 ± 4% to 54 ± 3% (P = 0.01), whereas the percentage of cells in S and G2 phases showed a statistically significant decrease from 43 ± 3% to 35 ± 4% (P = 0.02) and from 14 ± 1% to 11 ± 1% (P = 0.01), respectively. For HT29 cells, 48 hours of treatment with MN58b also caused a statistically significant increase in the percentage of cells in G1 phase from 60 ± 4% to 80 ± 2% (P = 0.007), and the percentage of cells in S and G2 phases decreased statistically significantly from 32 ± 4% to 17 ± 3% (P = 0.02) and from 8 ± 2% to 3 ± 1% (P = 0.04), respectively. On the other hand, 48 hours of treatment with the inactive analogue ACG20b had no statistically significant effect on the cell cycle distribution in both MDA-MB-231 and HT29 cells (P = 0.4; data not shown).

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In vitro 1H-MRS and 31P-MRS of cell extracts. [2]
MRS of carcinoma cells treated in vitro with MN58b was used to identify potential noninvasive markers of choline kinase inhibition. Figure 1B illustrates the 31P-MR spectra of control and 48-hour MN58b-treated MDA-MB-231 cells. Detailed analysis of 31P-MR spectra of control and MN58b-treated MDA-MB-231 cells at 4, 13, 16, 30, and 48 hours showed that MN58b treatment led to a statistically significant time-dependent drop in phosphocholine levels relative to controls which started as early as 4 hours (81 ± 6%, P = 0.04) and was down to 40 ± 2% (P = 0.00001) at 48 hours relative to controls (Fig. 1A). A statistically significant drop in phosphocholine (42 ± 13%, P = 0.03) relative to control was also detected following 48 hours incubation of HT29 cells with MN58b (Fig. 1C). Changes in other 31P-MR detectable metabolites (glycerophosphoethanolamine, glycerophosphocholine, and nucleotide triphosphosphates) were not statistically significant in either cell line (P > 0.05). It was not possible to accurately measure phosphoethanolamine levels in cell extracts due to the very small phosphoethanolamine signal in both cell lines.

31P-MR spectra of MDA-MB-231 and HT29 cells treated with ACG20b, the inactive analogue, were also investigated to verify that the 31P-MRS detected drop in phosphocholine is due to the inhibitory effect of MN58b on choline kinase. No statistically significant change in phosphocholine levels was observed in MDA-MB-231 (88 ± 11%, P = 0.2) or HT29 (88 ± 10%, P = 0.2) cells following 48 hours of treatment with ACG20b (Table 1; Fig. 1B and C). Changes in phosphocholine concentrations in MDA-MB-231 cells after a time course of treatment with MN58b are summarized in Table 1 and Fig. 1A.

1H-MR spectra of extracts of control and MN58b-treated MDA-MB-231 cells at 4, 13, 19, 30, and 48 hours were also investigated. A statistically significant time-dependent drop in the total choline content (choline + phosphocholine + glycerophosphocholine) was observed following 19 hours of incubation with MN58b (63 ± 17%, P = 0.03) relative to control, decreasing to 49 ± 9% (P = 0.002) at 48 hours following MN58b treatment. The decrease in total choline was similar to the drop in phosphocholine levels measured by 31P-MRS; hence, the decrease in the total choline is due to the drop in phosphocholine, whereas intracellular choline and glycerophosphocholine levels were not affected by MN58b treatment. Phosphocholine is required for the synthesis of phosphatidylcholine. Hence, to find out whether the drop in phosphocholine would cause a similar drop in the phosphatidylcholine levels, 1H-MR spectra of the lipid fractions of control and MN58b-treated MDA-MB-231 and HT29 cells were measured. No significant changes in phosphatidylcholine levels [using the N(CH3)3 peak resonating at 3.32 ppm] were detected following treatment of both cell lines with MN58b (P > 0.4; data not shown). These results are consistent with previous findings using NIH3T3 cells and human primary lymphocytes.

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Choline kinase activities in cell extracts and correlation to phosphocholine levels measured by MRS. [2]
14C choline labeling was used as a measure of choline kinase activity in MDA-MB-231 cells after a time course treatment with MN58b (Table 1; Fig. 1A). Significant correlation was found between phosphocholine concentrations (measured by MRS) and choline kinase activities (r2 = 0.95, P = 0.0008).

In HT29 and MDA-MB-231 xenografts, treatment with MN58b (4 mg/kg; i.p.; once daily; for 5 days; MF-1 nude mice) dramatically decreased phosphomonoesters. It was discovered that the phosphocholine level and bile.

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In vivo 1H-MRS and 31P-MRS of HT29 xenografts. [2]
In this study, MN58b inhibited tumor growth in HT29 xenografts by 70% (% treated versus control, % T/C), confirming previous findings. In vivo 1H-MR and 31P-MR spectra from a HT29 tumor pre-MN58b and post-MN58b treatment are shown in Fig. 2A, to D. Statistically significant decreases in the total choline concentration (P = 0.01) and phosphomonoester/total phosphorus signal ratio (P = 0.05) were also observed post-MN58b treatment (Table 2A). No statistically significant change in total choline concentration was observed in the control (vehicle treated) tumor group. A statistically significant increase in phosphomonoester/total phosphorus signal ratio (P = 0.04) was observed in the control group.

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In vivo 31P-MRS of MDA-MB-231 xenografts. [2]
Previous studies have shown statistically significant growth delays when MDA-MB-231 human breast carcinoma xenografts were treated with MN58b. Consistent with these results, we found that MN58b treatment for 5 days inhibited tumor growth by 85% (%T/C). In vivo 31P-MRS of the MDA-MB-231 tumors showed statistically significant decreases in the phosphomonoester/total phosphorus signal ratio (P = 0.05) following MN58b treatment (Table 2B). No statistically significant change was observed in the control (vehicle treated) tumor group.

Choline kinase activity [1]
Free 3H-choline was added to the reaction mix (MgCl2 10 mmol/L, KCl 100 mmol/L, ATP 500 μmol/L, and Tris pH 7.5 100 mmol/L), and its phosphorylation was determined from the amount of 3H-choline converted to 3H-phosphocholine using a modified Bligh and Dyer assay. The use of passive lysis buffer ensured enzymatic activity in the lysates before 3H-choline chloride was added. The reaction was stopped at 60 minutes by the addition of methanol/chloroform to effectively initiate the lipid extraction step. Phase extraction using tetraphenylborate then separated choline from phosphocholine, and the amount of 3H in each fraction was determined using a scintillation counter.

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Enzymatic assays of choline kinase activity in cell extracts. [2]
Following 4, 13, 19, 30, and 48 hours of treatment with 6 μmol/L MN58b, exponentially growing cells from three p60 plates (1 × 106) were lysed in 80 μL of lysis buffer [1.5 mmol/L MgCl2, 0.2 mmol/L EDTA, 0.3 mol/L NaCl, 25 mmol/L HEPES (pH 7.5), 20 mmol/L β-glycerophosphate, and 0.1% Triton X-100] to a final concentration of 3 μg/μL. From this, 90 μg of total protein were used per point as source of choline kinase in buffer containing 100 mmol/L Tris-HCl (pH 8), 100 mmol/L MgCl2, and 10 mmol/L ATP, as previously described.

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In vitro 1H-MRS and 31P-MRS of cell extracts. [2]
To obtain an MR spectrum, 1 × 107 to 2 × 107 cells in logarithmic phase were extracted from cell culture, as previously described. Briefly, cells were rinsed with ice-cold saline and fixed with 6 mL of ice-cold methanol. Cells were then scraped off the surface of the culture flask, collected into tubes, and vortexed for 30 seconds at room temperature to optimize phospholipid metabolite extraction from the ruptured cells. Chloroform (6 mL) was then added to each tube followed by an equal volume of deionized water. Following phase separation, the solvent in the upper methanol/water phase was removed using a freeze dryer, and the lipid metabolites were recovered by evaporation of chloroform to dryness, and samples were stored at −80°C until analysis. Before acquisition of the MRS spectra, the water-soluble metabolites were resuspended in deuterium oxide (D2O) for 1H-MRS or D2O with 10 mmol/L EDTA (pH 8.2) for 31P-MRS, and the lipid metabolites were resuspended in deuteriated chloroform for 1H-MRS. 1H-MRS and 1H-decoupled 31P-MRS spectra were acquired at room temperature on a 500 MHz Bruker spectrometer using a 30-degree flip angle, a 1-second relaxation delay, spectral width of 100 ppm, and 32 K data points for 31P; a 30-degree flip angle, a 1-second relaxation delay, spectral width of 12 ppm, and 32 K data points for 1H (lipids); and a 90-degree flip angle, a 1-second relaxation delay, spectral width of 12 ppm, 64 K data points, and HDO resonance suppression by presaturation for 1H (aqueous). Metabolite contents were determined by integration and normalized relative to the peak integral of an internal reference [0.15% 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt (water fractions) or 0.03% tetramethylsilane (lipid fractions) for 1H-MRS, and methylene diphosphonic acid (70 μL, 2 mmol/L) for 31P-MRS] and corrected for signal intensity saturation and the number of cells extracted per sample.

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In vitro 31P-MRS of tumor extracts. [2]
The freeze-clamped tumors were extracted in 6% perchloric acid, as previously described. Neutralized extracts were freeze-dried and reconstituted in 1 mL D2O, and the extracts (0.5 mL) were placed in 5-mm NMR tubes. For 1H-MRS, the water resonance was suppressed by using gated irradiation centered on the water frequency. 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt (50 μL, 5 mmol/L) was added to the samples for chemical shift calibration and quantification. Immediately before the MRS analysis, the pH of the samples was readjusted to 7 with perchloric acid or KOH. For 31P-MRS, which was carried out after the 1H-MRS study, EDTA (50 μL, 60 mmol/L) was added to each sample for chelation of metals ions, and methylene diphosphonic acid (50 μL, 5 mmol/L) was added to each sample for chemical shift calibration and quantification. The extract spectra for both the control and the treated animals were acquired under identical conditions.

Cell Viability Assay[1]
Cell Types: SK-PC-1, Suit2 008, IMIM-PC2, and RWP-1 - PC2 and RWP-1 cells) induced cells, a response that correlated with CHKα expression [1]. Cell
Tested Concentrations: 1 µM, 5 µM
Incubation Duration: 72 hrs (hours)
Experimental Results: Inhibition of cell growth.

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Apoptosis analysis [1]
Cell Types: SK -PC-1, Suit2 008, IMIM-PC2 and RWP-1 Cell
Tested Concentrations: 1 µM, 2 µM, 5 µM, 10 µM
Incubation Duration: 24 and 48 hrs (hours)
Experimental Results: Induction of apoptosis.

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Generation of MN58b-resistant cell lines [1]
To generate MN58b-resistant IMIM-PC-2 cells, MN58b was added starting at 0.1 μmol/L. Control IMIM-PC-2 cells were cultured without drug. MN58b concentration was increased by 50% weekly at each passage of the cells (split 1:3 when confluent); final concentration was 8 μmol/L. To generate gemcitabine-resistant PDAC cell lines, an incremental dose approach was used. The starting concentration was 35 nmol/L 2′-deoxy-2′,2′-difluorocytidine monohydrochloride. Gemcitabine concentration was maintained constant and was increased 1.5- to 2-fold at each cell passage. The final concentration was 250 nmol/L. Resistant cells were authenticated by STR profiling.

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Growth and viability assays [1]
Cells (5 × 104 per well) were seeded in triplicate in 6-well plates, trypsinized, and counted. To determine viability, cells (2 × 104 per well) were seeded in 24-well plates. After 24 hours, medium was removed and MN58b was added; after 72 hours, cells were fixed with 3% formaldehyde, washed twice with PBS, and incubated with 0.5% crystal violet in 25% methanol; crystal violet was eluted with 10% acetic acid and the OD590 nm was determined. To assess colony formation, cells (5 × 104 per well) were seeded in 6-well plates and medium was replaced 24 hours later with medium containing MN58b. After 72 hours, cells were processed as described earlier.

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Apoptosis assays [1]
Cells (5 × 104 per well) were seeded in 6-well plates in the presence of increasing concentrations of MN58b. After 24 and 48 hours, cells were washed with PBS, resuspended in Annexin V binding buffer, and incubated with APC-Annexin V in the dark for 15 to 20 minutes. DAPI was added for 15 minutes, and viability was assessed in a FACS Canto II flow cytometer. Results were quantified using FlowJo software.

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Drug synergy assays [1]
Cells (2 × 104 per well) were seeded in 24-well plates. After 24 hours, medium was removed and drugs (MN58b, gemcitabine, oxaliplatin, and 5-FU) were added for 72 hours, alone or in combination, at a range of doses according to the previously estimated IC50 for each drug. Viability was determined and the combination index (CI) values were calculated using the Chou and Talalay method with the Calcusyn software. A CI of 0.9–1.1 indicates an additive effect, a CI <0.9 indicates synergy, and a CI >1.1 indicates antagonism.

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Cell culture and treatment. [2]
MDA-MB-231 and HT29 cancer cell lines were cultured in DMEM supplemented with 10% FCS, 80 units/mL penicillin, and 80 μg/mL streptomycin at 37°C in 5% CO2. Cell growth inhibition (96 hours) for MDA-MB-231 cells with seeding density of 1 × 103 in 200 μL using 96-well plates, was measured by sulforhodamine B assay to assess IC50. MDA-MB-231 cells were treated with MN58b at pharmacologically active concentrations corresponding to 5 × IC50 (6 μmol/L) for 4, 13, 19, 30, and 48 hours or 6 μmol/L ACG20b (an inactive analogue) for 48 hours at 37°C, and HT29 cells were treated with 2.5 μmol/L MN58b or 2.5 μmol/L ACG20b for 48 hours at 37°C. The cells then underwent trypsinization and trypan blue exclusion assay. The effect of treatment on cell number was monitored by counting the number of attached cells in a treated flask and comparing that number with the number of attached cells in a control flask.[2]

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Cell cycle analysis. [2]
Cell cycle analysis of attached and detached control and treated cells was done on cells (1 × 106) fixed in 70% ethanol, treated with 100 μg/mL RNase A in citrate-buffered saline for 30 minutes at 37°C, and stained with 4 μg/mL propidium iodide, using an Elite Enhanced System Performance cell sorter at 488 nm. The cytometry data were analyzed using the WinMdi and Cylchred software[2].

Animal/Disease Models: MF-1 nude mice with HT29 or MDA-MB-231 cells [2]
Doses: 4 mg/kg
Route of Administration: intraperitoneal (ip) injection; base inhibitor activity [2]. one time/day; for 5 days.
Experimental Results: Phosphate monoesters diminished Dramatically.

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HT29 and MDA-MB-231 tumor xenograft models. [2]
MF-1 nude mice were injected s.c. in the flank with 0.2 mL of a suspension of HT29 human colon carcinoma cells (2.5 × 107/mL) or MDA-MB-231 human breast carcinoma cells (5 × 107/mL) that had been grown as a monolayer in cell culture. Tumor size was calculated by measuring the length, width, and depth of each tumor using calipers and by using the following formula: l × w × d × (π/6). Once an appropriate tumor size (approximate volume of 500 mm3) was established, mice were randomly divided into two groups: one group was treated with MN58b in saline at 4 mg/kg i.p. once a day for 5 days, and one group was treated with saline alone following the same regimen.

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In vivo 31P-MRS of HT29 and MDA-MB-231 tumor xenografts. [2]
Animals were anesthetized with a single i.p. injection of a Hypnovel/Hypnorm/water (1:1:2) mixture as previously described. Animals were placed in the bore of a Varian 4.7-T nuclear magnetic resonance (NMR) spectrometer and tumors were positioned in the center of a 12-mm two-turn 1H/31P surface coil. Image-selected in vivo spectroscopy–localized 31P-MR spectra of the tumors were obtained at 37°C, as previously described. Briefly, a gradient strength of up to 7.5 × 10−4 T/cm was applied with adiabatic pulses of 800 milliseconds, a 90-degree sincos excitation pulse, and a sech 180 inversion pulse, with a total repetition time of 3 seconds and 600 averages. 31P-MRS of the tumors was carried out before treatment (i.e., day 1) and 4 days after treatment (i.e., day 5). 31P-MR spectra were quantified using the VARiable PROjection program to determine precise chemical shifts and peak integrals as previously described. After the final 31P-MRS study, tumors were freeze clamped and stored at −80°C until analysis. The surface coils used to obtain the 31P-MRS signal from s.c. tumors in vivo were of nonuniform spatial sensitivity; thus, it is not possible to use an internal standard. Thus, the signal intensities observed in the in vivo 31P-MR spectra are expressed as ratios of metabolites.

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In vivo 1H-MRS of HT29 tumor xenografts. [2]
Anesthetized mice were placed in the MR system as described above. Voxels were selected from scout gradient echo images, and localized shimming yielded line widths of the order of 20 to 30 Hz. The PRESS localization method with water suppression was used to detect choline with a repetition time of 2 seconds, 64 transients, and echo times of 20, 68, 136, 272, and 408 milliseconds. For unsuppressed water, 16 transients were acquired with the same acquisition variables as above except with a lower receiver gain. After the final 1H-MRS study, tumors were freeze clamped and stored at −80°C until analysis. MRUI software was used for all spectral processing programs, including preprocessing, fitting, and quantification of peak areas of the observed metabolites. Eddy current correction was done by using the water FID to phase correct the metabolite signal point by point. This corrects the phase of the spectrum without any subjective bias. The last 100 data points were used to calculate root-mean-square noise and correct for direct current offsets. Using the choline and water peak areas as a function of echo time, choline and water T2 curves were fitted to a single exponential decay function and choline, and water intercepts (M0) and T2s were derived from these fits. The Levenberg-Marquardt algorithm was used for optimization. The choline concentration was calculated using tumor water as a reference assuming tumor tissue water was 80% (44 mol/L; ref. 44) and according to the equation.

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In vivo subcutaneous tumorigenic assay [1]
Suit2 028 CHKα-silenced (Sh-3 and Sh-5) and their nontarget counterparts (shNt) cells were grown in 6- to 8-week-old female BALB/c Nu/Nu mice. Cells (2 × 106, 100 μL in PBS) were injected subcutaneously, growth was monitored using an electronic caliper, and volumes calculated using the formula (L × W2 × 0.5). Mice were housed in IVC cages.

[1]. Choline Kinase Alpha (CHKα) as a Therapeutic Target in Pancreatic Ductal Adenocarcinoma: Expression, Predictive Value, and Sensitivity to Inhibitors. Mol Cancer Ther. 2016 Feb;15(2):323-33.

[2]. Noninvasive magnetic resonance spectroscopic pharmacodynamic markers of the choline kinase inhibitor MN58b in human carcinoma models. Cancer Res. 2006 Jan 1;66(1):427-34.

Choline kinase α (CHKα) plays a crucial role in the regulation of membrane phospholipid synthesis and has oncogenic properties in vitro. We have analyzed the expression of CHKα in cell lines derived from pancreatic ductal adenocarcinoma (PDAC) and have found increased CHKα expression, associated with differentiation. CHKα protein expression was directly correlated with sensitivity to MN58b, a CHKα inhibitor that reduced cell growth through the induction of apoptosis. Accordingly, CHKα knockdown led to reduced drug sensitivity. In addition, we found that gemcitabine-resistant PDAC cells displayed enhanced sensitivity to CHKα inhibition and, in vitro, MN58b had additive or synergistic effects with gemcitabine, 5-fluorouracil, and oxaliplatin, three active drugs in the treatment of PDAC. Using tissue microarrays, CHKα was found to be overexpressed in 90% of pancreatic tumors. While cytoplasmic CHKα did not relate to survival, nuclear CHKα distribution was observed in 43% of samples and was associated with longer survival, especially among patients with well/moderately differentiated tumors. To identify the mechanisms involved in resistance to CHKα inhibitors, we cultured IMIM-PC-2 cells with increasingly higher concentrations of MN58b and isolated a subline with a 30-fold higher IC50. RNA-Seq analysis identified upregulation of ABCB1 and ABCB4 multidrug resistance transporters, and functional studies confirmed that their upregulation is the main mechanism involved in resistance. Overall, our findings support the notion that CHKα inhibition merits further attention as a therapeutic option in patients with PDAC and that expression levels may predict response. [1]

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MN58b is a novel anticancer drug that inhibits choline kinase, resulting in inhibition of phosphocholine synthesis. The aim of this work was to develop a noninvasive and robust pharmacodynamic biomarker for target inhibition and, potentially, tumor response following MN58b treatment. Human HT29 (colon) and MDA-MB-231 (breast) carcinoma cells were examined by proton (1H) and phosphorus (31P) magnetic resonance spectroscopy (MRS) before and after treatment with MN58b both in culture and in xenografts. An in vitro time course study of MN58b treatment was also carried out in MDA-MB-231 cells. In addition, enzymatic assays of choline kinase activity in cells were done. A decrease in phosphocholine and total choline levels (P < 0.05) was observed in vitro in both cell lines after MN58b treatment, whereas the inactive analogue ACG20b had no effect. In MDA-MB-231 cells, phosphocholine fell significantly as early as 4 hours following MN58b treatment, whereas a drop in cell number was observed at 48 hours. Significant correlation was also found between phosphocholine levels (measured by MRS) and choline kinase activities (r2 = 0.95, P = 0.0008) following MN58b treatment. Phosphomonoesters also decreased significantly (P < 0.05) in both HT29 and MDA-MB-231 xenografts with no significant changes in controls. 31P-MRS and 1H-MRS of tumor extracts showed a significant decrease in phosphocholine (P < or = 0.05). Inhibition of choline kinase by MN58b resulted in altered phospholipid metabolism both in cultured tumor cells and in vivo. Phosphocholine levels were found to correlate with choline kinase activities. The decrease in phosphocholine, total choline, and phosphomonoesters may have potential as noninvasive pharmacodynamic biomarkers for determining tumor response following treatment with choline kinase inhibitors. [2]

C32H40BR2N4

640.4948

640.159

C, 60.01; H, 6.30; Br, 24.95; N, 8.75

203192-01-2

203192-01-2 (bromide);730930-74-2 (cation);

52952144

White to off-white solid powder

0

4

11

38

523

0

[Br-].[Br-].[N+]1(C([H])=C([H])C(=C([H])C=1[H])N(C([H])([H])[H])C([H])([H])[H])C([H])([H])C1C([H])=C([H])C(=C([H])C=1[H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C1C([H])=C([H])C(=C([H])C=1[H])C([H])([H])[N+]1C([H])=C([H])C(=C([H])C=1[H])N(C([H])([H])[H])C([H])([H])[H]

YLHINOUDZGYMNO-UHFFFAOYSA-L

InChI=1S/C32H40N4.2BrH/c1-33(2)31-17-21-35(22-18-31)25-29-13-9-27(10-14-29)7-5-6-8-28-11-15-30(16-12-28)26-36-23-19-32(20-24-36)34(3)4;;/h9-24H,5-8,25-26H2,1-4H3;2*1H/q+2;;/p-2

1-[[4-[4-[4-[[4-(dimethylamino)pyridin-1-ium-1-yl]methyl]phenyl]butyl]phenyl]methyl]-N,N-dimethylpyridin-1-ium-4-amine;dibromide

MN58b bromide; MN 58b; MN58b; 203192-01-2; 1-[[4-[4-[4-[[4-(dimethylamino)pyridin-1-ium-1-yl]methyl]phenyl]butyl]phenyl]methyl]-N,N-dimethylpyridin-1-ium-4-amine;dibromide; 1,1'-((Butane-1,4-diylbis(4,1-phenylene))bis(methylene))bis(4-(dimethylamino)pyridin-1-ium) bromide; Pyridinium, 1,1'-[1,4-butanediylbis(4,1-phenylenemethylene)]bis[4-(dimethylamino)-, bromide (1:2); 4-(dimethylamino)-1-({4-[4-(4-{[4-(dimethylamino)pyridin-1-ium-1-yl]methyl}phenyl)butyl]phenyl}methyl)pyridin-1-ium dibromide; CHEMBL1771545; MN58b?; MN 58b; MN-58b

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~14.7 mg/mL (~23 mM)

Solubility in Formulation 1: ≥ 1.47 mg/mL (2.30 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 14.7 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 1.47 mg/mL (2.30 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 14.7 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)