31P and 1H MRS of Androgen-Independent and Androgen-Dependent Prostate Cancer Xenografts: Lonidamine Selectively Decreases Tumor Intracellular pH, Bioenergetics and Increases Lactate
Kavindra Nath1, David Nelson1, Dennis Leeper2, and Jerry Glickson1

1University of Pennsylvania, Philadelphia, PA, United States, 2Thomas Jefferson University, Philadelphia, PA, United States

Synopsis

Lonidamine (LND) effects were measured in vivo by 31P and 1H MRS in androgen-independent (PC3) and androgen-dependent (LNCaP) prostate cancer xenografts indicating a sustained and tumor-selective decrease in intracellular pH (pHi) and extracellular pH (pHe), and decrease in tumor bioenergetics (βNTP/Pi) by 75.0 % and 79.0 % in PC3 and LNCaP, respectively, relative to the baseline levels. Steady-state levels of tumor lactate were significantly increased ~ 2 fold at 60 min. post-LND. The decline of pHi, pHe, bioenergetics and increase in lactate produced increased therapeutic efficacy when LND was combined with other therapeutic interventions (chemotherapy, radiation, and hyperthermia).

Introduction

Specifically, we seek to employ the natural tendency of tumors to convert glucose to lactate as a method for selective intracellular tumor acidification, which has been reported to potentiate tumor response to radiation (1), hyperthermia (2) as well as to chemotherapy with N-mustards (3, 4), doxorubicin (5) and alkylating agents. This study monitors intracellular pH (pHi), extracellular pH (pHe), bioenergetics (βNTP/Pi) and lactate in androgen-independent (PC3) and androgen-dependent (LNCaP) prostate cancer xenografts by 31P and 1H magnetic resonance spectroscopy (MRS) following administration of lonidamine (LND), a putative inhibitor of transmembrane monocarboxylate transporters (MCTs) (unpublished data), the mitochondrial pyruvate carrier (unpublished data) and complex II of electron transport chain (6). These findings point to the potential utility of radiation, hyperthermia and chemotherapeutic drugs in combination with LND in the treatment of disseminated prostate cancer.

Material and Methods

PC3 prostate cancer cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2mM L-glutamine, and 1% penicillin-streptomycin. 7×106 PC3 cells were inoculated subcutaneously (s.c.) in each mouse (n=5) as a 0.1 mL suspension. LNCaP cells were grown in RPMI 1640 with 10% fetal bovine serum and 0.5% penicillin/streptomycin. 5×106 cells in a mixture of 75 μl matrigel and 75 μl of RPMI 1640 medium were inoculated s.c. into the right flank of the nude mouse (n=5). Prostate cancer xenografts were allowed to grow until they reached 7-10 mm in diameter along the longest axis of the tumor. The pHi (n=5), extracellular pH (pHe) (n=5), βNTP/Pi (n=5) and steady-state levels of tumor lactate (n=5) were measured in PC3 and LNCaP xenografts by 31P MRS (pH and bioenergetics) and 1H MRS (Hadamard-selective multiple quantum coherence transfer pulse sequence), respectively, as described in our previous publications (3-5). Analysis of variance with Tukey multiple comparisons was used for statistical analysis (SPSS 16). The data on pHi, pHe, bioenergetics and lactate at various time points following LND administration were compared by ANOVA and t test analysis.

Results

In vivo 31P MRS (Fig. 1) demonstrates that PC3 and LNCaP prostate cancer xenografts in immunosuppressed mice treated with the MCT inhibitor LND exhibit a sustained and tumor-selective decrease in pHi from 6.94 ± 0.02 to 6.49 ± 0.05 (p = 0.02), pHe from 7.06 ± 0.03 to 6.72 ± 0.08 (p = 0.08) (Fig. 2 A) and 6.90 ± 0.03 to 6.45 ± 0.05 (p = 0.05), pHe from 7.0 ± 0.03 to 6.69 ± 0.06 (p = 1.0) (Fig. 2 B), respectively. Tumor bioenergetics (βNTP/Pi) decreased by 75.0 ± 0.12% (p = 0.01) and 79.0 ± 0.18% (p = 0.00) in PC3 and LNCaP (Fig. 2C), respectively, relative to the baseline level immediately prior to LND administration. Steady-state levels of tumor lactate (intracellular plus extracellular) were monitored by 1H MRS with the Hadamard-selective multiple quantum coherence transfer pulse sequence in PC3 (Fig. 3A, C) and LNCaP (Fig. 3B, C) tumors following LND administration at time zero. The lactate intensity, which peaked at around 40 min in both cell type, PC3 (p = 0.03) and LNCaP (p = 0.02) remained stable for 120 min (p = 0.03) in PC3 and 100 min (p = 0.02) in LNCaP post- LND administration, and then decreased monotonically (Figs 3 A, B, C).

Discussion

The 31P MR spectra clearly show that LND leads to intracellular acidification and depression of the bioenergetics of the prostate cancer xenograft in vivo; these parameters are critical indices for tumor thermosensitization and/or for improving tumor response to antineoplastic agents. LND appears to inhibit the MCT on the plasma membrane and may also block the mitochondrial pyruvate carrier thereby impeding the delivery of pyruvate to produce acetyl-CoA for the TCA (tricarboxylic acid) cycle (unpublished data). Thus, LND is very attractive because it may simultaneously cause selective tumor acidification and tumor de-energization. While LND clearly inhibits export of lactate from human DB-1 melanoma and MCF-7 breast cancer and human 9L gliomas in rats, it is not clear if it is inhibiting transport of pyruvate into mitochondria as α-cyano-4-hydroxycinnamate (CHC) does (7-9). However, the similar effect of CHC and LND on the bioenergetics of DB-1 melanomas (3, 10) and prostate cancer here strongly suggest that it is. Therefore, the decline of bioenergetics that was evident both from the decrease in βNTP/Pi and from direct monitoring of NTP by 31P MRS vs. time in each animal could be explained by a profound decrease in mitochondrial metabolism following LND administration.

Acknowledgements

NIH grants R01-CA129544 and R01-CA172820. Jeff Roman and Kevin Muriuki are acknowledged for their help to grow PC3 and LNCaP cells.

References

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(3). Nath K, Nelson DS, Ho AM, Lee SC, Darpolor MM, Pickup S, Zhou R, Heitjan DF, Leeper DB, Glickson JD. 31P and 1H MRS of DB-1 melanoma xenografts: lonidamine selectively decreases tumor intracellular pH and energy status and sensitizes tumors to melphalan. NMR Biomed. 2013 Jan;26(1):98-105. doi: 10.1002/nbm.2824. Epub 2012 Jun 29.

(4). Nath K, Nelson DS, Heitjan DF, Zhou R, Leeper DB, Glickson JD. Effects of hyperglycemia on lonidamine-induced acidification and de-energization of human melanoma xenografts and sensitization to melphalan. NMR Biomed. 2015 Mar;28(3):395-403.

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Figures

Figure 1. In vivo localized 31P MRS of PC3 (left) and LNCaP (right) prostate cancer xenograft grown subcutaneously in nude mice (lower) baseline- and (upper) 180 min. post LND administration (100 mg/kg, i.p.). Decrease in βNTP levels and the corresponding increase in Pi following LND administration (Spectrum B) indicating impaired energy metabolism.

Figure 2. The intracellular pH, extracellular pH profile of PC3 (n=5) (A), LNCaP (n=5) (B), the changes of bioenergetics (βNTP/Pi) (ratio of peak area) relative to baseline as a function of time of PC3 and LNCaP (C) prostate cancer xenografts in response to LND (100 mg/kg; i.p.) administration at time zero.

Figure 3. Spectra show the effect of LND (100 mg/kg; i.p.) on tumor lactate production in PC3 (A) and LNCaP (B). Area under the curve was compared to baseline at each time points and was normalized to baseline levels (C). LND was administered at time zero.



Proc. Intl. Soc. Mag. Reson. Med. 24 (2016)
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