Abstract

The goal of this project is to treat the cancer patient by reinstating the normal immunological balance between the patient and the cancer. In order for this to happen, communication with cancer is of paramount importance. We have explored the possibility of interacting with cancer through electrical charges and by bringing the immune system back to normalcy. We hypothesized that the immune system in this state would drive cancer into apoptosis. The chemical molecule used to communicate with the cancer caused a 48% reduction in the tumor’s size after merely four days of injections. The experimental model used was the 4T1 human breast cancer analog in vivo and the MDA-MB-231 cell line in vitro.

 

Keywords: cancer microenvironment, apoptosis, protons, pH, glucose, immune therapy

 

Introduction 

Quickly growing malignant tumors are often distinguished by higher glycolysis rates compared with normal tissues 1, 2, 3. This metabolic glycolytic shift of cancer cells rapidly causes the accumulation of protons (H+) in the extracellular space thus increasing the acidity around the tumor 4, 5, 6. This peritumoral acidity is not only involved in driving invasion and metastasis but also in reducing radiotherapy and chemotherapy efficacy 7, 8. A normal tumor pH extracellularly can flux between 6.2-6.8, compared to 7.2-7.4 in regard to normal cells. 

 

On the contrary, inside the cancer cell, the increased proton production - from the altered metabolic profile of the cell - is followed by increased proton secretion 9, 10. The easier a cancer cell secretes protons to the extracellular space, the more malignant it becomes 11. An increased pH intracellularly (pHi) leads to increased proliferation and angiogenesis which in combination with the decreased pH extracellularly (pHe) leads to increased survival and metastasis. 

 

The metabolic shift to increased glycolysis, even under aerobic conditions, demands higher quantities of glucose 12, 13. GLUT transporters, the membrane proteins that facilitate glucose transport inside the cell, are often over expressed in malignant tumors in order for the cancer cells to gain access to more glucose 14. This increased glucose uptake of the cancer cells is routinely used in the Positron Emission Tomography (PET) scan to visualize tumors.

 

Vision 

Based on the new metabolic order of cancer that results in the reversed proton (+) gradient across the cell membrane 15, 16, 17, it was hypothesized that charges - ionic charges - may be the chemical means of interacting with cancer. Protons (hydrogen ions, H+) are the smallest and the most reactive ions in our bodies. Since most solid tumors - glycolytic or non-glycolytic 18, 19 - have higher acidity in their periphery 20, the cancer language used to convey the message should be of an electrical, ionic nature. This is not a message of killing the cancer or inhibiting one of its chemical pathways; instead, it is an effort focused on restoring the immunological balance of the tumor-host interface through proton neutralization. By disrupting the new cancer-induced pH homeostasis, an attempt was made to reinstate the lost immunological equilibrium. Practically, these efforts were focused on driving the malignant cells into apoptosis by reestablishing normal pH levels.

 

The cancer balance

Somewhere in the body, for some reason (beyond the scope of this investigation), there is a source of exceedingly high number of protons which create an immunological imbalance of immense clinical proportions 20. This proton source transforms the local environment into a new entity that does not respond to the usual growth suppressor signals. The newly created protons tip the local immunological balance into favoring the new entity-i.e., the cancer 21, 22. Subsequently, the cancer, following its own rules, slips into uncontrollable proliferation. 

 

The normal balance

It was determined that neutralizing the proton should be employed when bringing the cancer environment back to its normal order. This would be a new chemical compound which could neutralize the newly formed protons responsible for the new metabolic order. The goal would not be to kill the cancer cells but rather to instruct them to return to their previous state, which is tightly regulated by immunological rules. The byproduct of this strategy is cancer apoptosis. 

 

Design of the molecule

This molecule should have three parts: one part for locating the cancer environment, one for conducting proton neutralization and one to connect these two together and confirm that they work independently. The first part should direct the molecule to the cancer environment. A greater penetration into the cancerous tumor is an ideal addition to this first phase. The second part, the proton neutralizer, should be inactive at normal pH levels and only abrogate protons where it encounters an acidic milieu. This would assure that it does not harm cells which are at normal pH levels and would work only when it reaches a locale of extra acidity. Finally, there should be a link present to assure that these two parts work independently. 

 

Parts of the molecule

The great affinity of cancer cells to glucose is a well established fact 24. The glucose molecule, in addition, has the ability to penetrate deep into the cancer mass 24. Based on this understanding, we know concretely that glucose has the unique skill of reaching cancer’s acidic microenvironment (based on PET Scans 25). The chemical that we will introduce for the neutralization of the cancer protons will be an amine (the NH2 molecule) which is non-toxic to normal cells. The remaining link will be a simple polyethylene glycol system (PEG), which will improve both the safety and efficiency of this process.

 

Rationale of function

Glucose is able to penetrate glycolytic solid tumors easier and more deeply than oxygen. It is determined that the glucose element will open the cancer mass and allow the whole molecule to be dragged deeper within. Along the way, from the extracellular space to inside the cell, it is believed that the amine will be protonated. The acidic environment outside the cancer cells will force the amine to pick up just one proton and to become NH3. We believe that this protonation is what will eventually lead to the disruption of the cancer pH homeostasis by acid loading the cytosol, de novo 26. The cancer expels the protons out of the cytosol, this compound brings them back inside the cell 27. We assume that its mechanism of action resembles that of the NH4Cl intracellular acid loading. The NH4 leaves the cell as uncharged NH3,  thereby leaving protons behind and causing a cytosolic acid loading. Summarizing, the goal is the disruption of the cancer-induced pH homeostasis, which will lead the cancer cell into apoptosis. Glucose is the best vehicle for such a job because it brings the whole molecule inside the cell [glutamine 28, 29 or fatty acids may be other preferred vehicles (future development)]. It is not the buffering capacity of the molecule -per se- that this approach is after but rather the disturbance, the disruption of the cancer pH. The slightest variation in the pHi/pHe ratio, as little as 0.1 pH units, drastically alters the proliferation-migration-invasion profile of cancer. This cancer therapeutic approach is a systems biology approach and not a reductionist attempt to kill the cancer cells.

 

Attempts in the past with the glucose molecule

In the early 1950s a new molecule was developed to fight cancer. The concept at that time was to stop glycolysis; the compound formed from this process was deoxyglucose (DG). The glucose used in this compound had the [OH] in the 2-position and was replaced with a hydrogen atom. Unfortunately, the DG stopped glycolysis everywhere - it also blocked it in the brain - and it could not be used as a drug 31 (twenty years later it was transformed into an excellent diagnostic-imaging tool, F-DG, which is used in PET scans). On the contrary, the recommended molecule maintains the glucose part almost intact and neither stops nor alters glycolysis. We have observed the agent being metabolized over time by the cancer cells in vitro (unpublished data) that led us to believe that this compound does not interfere with the glycolytic cycle of the cell and that the effect that exerts on the cancer cells is not due to altering the glucose metabolism.

 

Concluding remarks - Highlights

  • It is not the cancer per se that is intended to be eliminated, cancer cells are still considered to be “self” cells that have stopped following the physiological rules.

  • Reinstating normalcy, the cancer cells are driven into apoptosis and this is done by disrupting the cancer-induced pH gradient intra-extracellularly.

  • By following this line of thinking, it is seen that cancer creates protons. These protons, in turn, evade immunological attack. The response is to neutralize these protons, forcing cancer to return to apoptosis (apoptosis is cancer’s own normalcy). 

  • Could this simplistic logic give us another weapon in the immunotherapy arsenal against cancer? 30

 

Questions need to be answered

  • Are protons the words of cancer’s language? 

  • Should our therapeutic roles be more about adjusting equilibriums rather than killing cells?

  • And lastly, and even more importantly, could we finally be able to influence the immune system and tip the immunological balance in favor of normalcy and not cancer?

 

The Experiment in vivo

 

The purpose: to assess the anti-tumor activity of the compound IF-002 (GlcPEG) on a model of mouse 4T1 breast tumor in Balb/C mice. 

 

Animal and Facility: healthy female Balb/c (5-6 weeks old), purchased from Harlan Laboratories, were allowed for acclimation for 3 days in a specific-pathogen-free animal facility. Animal housing, handling and procedures were followed to those protocols and guidelines approved by the lab’s Institutional Animal Care and Use Committee (IACUC). 

 

In vivo Syngeneic Tumor Study Design: Murine 4T1 breast tumor cells were kept in RPMI-1640 medium containing 10% FBS and 1% Pen/Strip. On the day of inoculation, the cells were harvested according to standard protocol (Beth Pulaski) and re-suspended in PBS at the cell concentration of 2.5x105 cells/ml. Each animal was injected 0.1 ml of the cell solution into the right flank of Balb/c mice following the SOP. On day 8 post-inoculation, all tumor volumes were measured and the animals were divided into two groups, control and treated. 

 

Study design: to deliver a predetermined dose of the compound GlcPEG IV (intravenously) for 5 days.

 

Study objective: to observe clinically the reduction of the size of the tumors (our “biomarker”).

 

Treatment: the tested compound was prepared right before dosing on day 8 after 4T1 inoculation. Each mouse received a predetermined dose of either the compound or saline intravenously daily for 5 consecutive days. The mice tumor volumes and mice body weights were monitored every other day along with other general behaviors factors.

 

Endpoints: animals were euthanized if one of the following conditions was identified:

  • Its neoplasm reached the predetermined endpoint size of 1000 mm3 

  • The animal became moribund

  • Body weight lost more than 20% from its original weight

 

Results: in the study, the 4T1 tumor growth was similar as our previous historic data. The control tumors were doubled about every 2-3 days. However, the tumors in the treated group by the compound grew slowly. That means that the compound can inhibit 4T1 tumor growth during the treatment (from day 8 to day 12). In fact, the maximal tumor growth inhibition index (T/C ratio) was around 48% (day 10). The inhibitory effect on the tumor growth by the compound was tolerated for the tumor and no toxic effects were observed in the mice.

Histology slides that show on the left the control without treatment and on the right with treatment. Gross size reduction was our goal.

Measuring the gross size reduction of the tumor with calipers

The Experiment in vitro

 

The purpose: to assess the apoptotic effect of the compound GLc-PEG.

 

The cells: MDA-MB-231 human breast cancer cell line.

 

The design: to treat some MDA-MB-231s with GLc-PEG and some 231s with saline (control) for an hour.

 

The results: 

Cytology image of treated MDA-MB-231 cells 

Cytology image of untreated MDA-MB-231 cells 

Cytology image analysis of treated and untreated MDA-MB-231 cells 

 

GLcPEG induced apoptosis is typified by:

  • condensation and margination of the chromatin

  • formation of crescents 

  • cell shrinkage 

  • increased staining 

  • budding

  • cytoplasmic vacuolization 

  • nuclear fragmentation     

Chromatin condensation   

Formation of crescents

   Shrinkage 

Compared to untreated cell

Nuclear fragmentation

Budding

Vacuolization

In a nutshell, the whole therapeutic approach

Cancer creates protons

Protons create immunological evasion and chemoresistance

Evasion establishes cancer's normalcy-host's death

Protons are neutralized

Proton neutralization reinstates normal pH levels

Normal pH levels establish host's normalcy-cancer's death

References

  1. Ward PS, Thompson CB. Metabolic reprogramming: a cancer hallmark even Warburg did not anticipate. Cancer Cell. 2012;21(3):297–308. 

  2. Vander Heiden MG, Cantley LC, Thompson CB. Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science. 2009;324(5930):1029–1033. 

  3. P. Vaupel, O. Thews, D.K. Kellether, M. Hoeckel. Current status of knowledge and critical issues in tumor oxygenation—results from 25 years research in tumor pathophysiology. Adv. Exp. Med. Biol. 454 (1998) 591–602. 

  4. Gillies RJ, Liu Z, Bhujwalla Z. 31P-MRS measurements of extracellular pH of tumors using 3-aminopropylphosphonate. Am J Physiol. 1994 Jul;267(1 Pt 1):C195–C203.

  5. Chiche, J., Brahimi-Horn, M. C. and Pouyssegur, J. (2010) Tumour hypoxia induces a metabolic shift causing acidosis: a common feature in cancer. J. Cell. Mol. Med., 14: 771–794.

  6. Gatenby, R. A., Gawlinski, E. T., Gmitro, A. F., Kaylor, B., and Gillies, R. J. Acid-mediated tumor invasion: a multidisciplinary study. (2006) Cancer Res. 66, 5216 –5223

  7. Reshkin SJ et al. 2000 Na+/H+ exchanger-dependent intracellular alkalinization is an early event in malignant transformation and plays an essential role in the development of subsequent transformation-associated phenotypes. FASEB J. 14, 2185-2197

  8. Gillies RJ et al. 1990 Tumorigenic 3T3 cells maintain an alkaline intracellular pH under physiological conditions. Proc. Natl. Acad. Sci. USA 87, 7414-7418

  9. Pouyssegur J, Sardet C, Franchi A, L'Allemain G, Paris S. A specific mutation abolishing Na+/H+ antiport activity in hamster fibroblasts precludes growth at neutral and acidic pH. Proc. Natl. Acad. Sci. USA. 1984 Aug;81(15):4833–4837.37

  10. Boron F. W.  Regulation of intracellular pH. Adv. Physiol. Edu. Dec. 2004 Vol. 28 no. 4, 160-179 

  11. Bradley A. Webb, Michael Chimenti, Matthew P. Jacobson & Diane L. Barber Dysregulated pH: a perfect storm for cancer progression. Nature Reviews Cancer 11, 671-677 (September 2011)

  12. O. Warburg On the origin of cancer cells. Science 123 (1956) 309–314

  13. O. Warburg, et al. The metabolism of tumors in the body. J. Gen. Physiol., 8 (1927), pp. 519–530

  14. Macheda ML, Rogers S, Best JD. Molecular and cellular regulation of glucose transporter (GLUT) proteins in cancer. J Cell Physiol. 2005; 202: 654–662. 

  15. Gerweck, L. E., Kozin, S. V., and Stocks, S. J. The pH partition theory predicts the accumulation and toxicity of doxorubicin in normal and low-pH-adapted cells. Br. J. Cancer, 79: 838–842, 1999. 

  16. Ebbesen P et al. Taking advantage of tumor cell adaptations to hypoxia for developing new tumor markers and treatment strategies. J. Enzyme Inhib. Med. Chem. 24 (Suppl. 1), 1-39 (2009)

  17. Kato Y, Ozawa S, Miyamoto C, Maehata Y, Suzuki A, Maeda T, Baba Y. Acidic extracellular microenvironment and cancer. Cancer Cell Int. 2013 Sep 3;13(1):89

  18. Helmlinger G, Sckell A, Dellian M, Forbes NS, Jain RK. 2002 Acid production in glycolysis-impaired tumors provides new insight into tumor metabolism. Clin. Cancer Res. 8, 1284-1291

  19. Newell K, Franchi A, Pouyssegur J, Tannock l. 1993 Studies with glycolysis-deficient cells suggest that production of lactic acid is not the cause of tumor acidity. Proc. Natl. Acad. Sci. USA 90, 1127-1131.

  20. Vaupel P, Kallinowski F, Okunieff P. Blood flow, oxygen and nutrient supply, and metabolic microenvironment of human tumors: a review. Cancer Res 1989; 49: 6449–6465.

  21. Swietach P, Vaughan-Jones RD, Harris AL, Hulikova A. The chemistry, physiology and pathology of pH in cancer. Philos T Roy Soc B: Biological Sciences. 2014;369 (1638):20130099.  

  22. Yabu M, et al. IL-23-dependent and -independent enhancement pathways of IL-17A production by lactic acid. Int Immunol. 2011;23(1):29–41. 

  23. Shime H, et al. Tumor-secreted lactic acid promotes IL-23/IL-17 proinflammatory pathway. J Immunol. 2008;180(11):7175–7183.

  24. Mathupala SP et al. 2010 The pivotal roles of mitochondria in cancer: Warburg and beyond and encouraging prospects for effective therapies. BBA 1797 1225-1230

  25. RA Gatenby, RJ Gillies 2004 Why do cancers have high aerobic glycolysis. Nat Rev Cancer 4 (11), 891-899 

  26. Park HJ, Lyons JC, Ohtsubo T, Song CW. Acidic environment causes apoptosis by increasing caspase activity. Br. J. Cancer 80. 1892-1897 (1999).

  27. Shrode L, Tapper H, Grinstein S. Role of intracellular pH in proliferation, transformation and apoptosis. J. Bioenerg. Biomembr. 29. 393-399 (1997).

  28. Son et al. Glutamine supports pancreatic cancer growth through a KRAS-regulated metabolic pathway. Nature. 2013;496(7443):101–105. 

  29. DeBerardinis RJ, Cheng T. Q’s next: the diverse functions of glutamine in metabolism, cell biology and cancer. Oncogene. 2010;29(3):313–324. 

  30. Zhao Y, Butler EB, Tan M. Targeting cellular metabolism to improve cancer therapeutics. Cell Death Dis 2013; 4: e532

  31. Gambhir SS 2002 Molecular imaging of cancer with positron emission tomography. Nat Rev Cancer 2 683-693

 

Acknowledgements 

I would like to thank Ian F. Robey, research assistant professor at the University of Arizona, for his excellent job with the in vitro experiments and the photos posted earlier in the article.

I would also like to thank Christos Douvris, assistant professor of Chemistry at the McNeese University, for his expertise and important contributions to my research at a very early stage in its development. 

I would finally like to express my thanks to Matthew Bemis for his assistance in editing this text.