SAMEER KALGHATGI

           

This page gives a brief overview of Melanoma Cancer, Cancer Therapy, Apoptosis and my research on Induction of Apoptosis in Melanoma Cancer cells by Non-Thermal Atmospheric Pressure Dielectric Barrier Discharge Plasma.

Melanoma Cancer

Melanoma is a malignant tumor of melanocytes which are found predominantly in skin but also in the bowel and the eye. It is one of the rarer types of skin cancer but causes the majority of skin cancer related deaths. Malignant melanoma is a serious type of skin cancer. It is due to uncontrolled growth of pigment cells, called melanocytes (1, 2). Despite many years of intensive laboratory and clinical research, the sole effective cure is surgical resection of the primary tumor before it achieves a thickness greater than 1 mm. Around 160,000 new cases of melanoma are diagnosed worldwide each year, and it is more frequent in males and Caucasians (3). It is more common in Caucasian populations living in sunny climates than in other groups (4). According to a WHO report about 48,000 melanoma related deaths occur worldwide per year (5). Malignant melanoma accounts for 75 percent of all deaths associated with skin cancer (6).

Non-Thermal Plasma for Cancer Therapy

The treatment for melanoma cancer includes surgical removal of the tumor, adjuvant treatment, chemo- and immunotherapy, or radiation therapy. All these means of treatment of melanoma skin cancer have some serious and unpleasant side effects. Chemotherapy leads to hair loss, fatigue, anemia, prone to infection etc. Radiation also leads to fatigue, loss of appetite, increased rate of infections etc. Further these treatment modalities are expensive and need extensive equipment and drugs.

In view of these potential issues related to traditional methods of cancer therapy, A. J. Drexel Plasma Institute initiated a study to develop Non-Thermal Plasma as a low cost, portable, novel clinical tool for a wide range of clinical applications. Non-thermal plasma can kill bacteria or induce apoptosis in malignant cells (4, 5, 6, 7). It can be applied in sub-lethal doses to elicit specific biological effects, including gene transfection (8-10), cell detachment (11-14), wound healing (5, 15-17) and blood coagulation (18). Non-thermal plasma can even have selective effects. In recent studies of plasma blood coagulation (18) and bacteria deactivation (4, 5), plasma did not demonstrate measurable toxicity in the surrounding living tissue (5, 10).

The operating principle of this plasma discharge is similar to the Dielectric Barrier Discharge (DBD) introduced by Siemens in 1859 (19). DBD occurs at atmospheric pressure in air or other gases when high voltage of sinusoidal waveform or short duration pulses is applied between two electrodes, with at least one electrode being insulated (20). The insulator prevents current build-up between the electrodes, creating electrically safe plasma without substantial gas heating. This approach allows direct treatment of melanoma skin cancer without the side effects observed after chemo or radiation therapy or thermal damage observed in more traditional thermal plasmas (21)

Our floating electrode dielectric barrier discharge (Plasma) system is constructed similarly to conventional dielectric barrier discharges and is inherently non-thermal – it is able to operate at room temperature and pressure.  Plasma operates under conditions where one of the electrodes is a dielectric-protected powered electrode and the second active electrode is a human or animal skin or organ.  Without human or animal skin or tissue surface present, a discharge does not ignite. Our lab has been working with non-thermal atmospheric plasma discharges for use on living tissues.  To date there has been no investigation of the interaction between non-thermal atmospheric plasma discharges and the induction of apoptosis in living cells.  This information is critical if this is going to be a viable treatment strategy as a selective cytotoxic cancer therapy.

Non-Thermal Plasma Initiated Apoptosis in Cancer Cells

The ability of a cell to self-regulate is a vital function in higher organisms allowing for appropriate growth, development, and death at the necessary times.  Apoptosis, or programmed cell death, is a critical element of this self-regulation.    The non-functioning of a tumor-suppressor gene that facilitates apoptosis, or the over expression of an anti-apoptotic protein are both important pathways in cancer development.  Many anti cancer therapies are aimed at modulating these factors.  Researchers are working with various bioactive agents in an attempt to target various components of the apoptotic pathways.  Many of these approaches however still remain in the preclinical development secondary to low efficacy and drug resistance.  Our current research seeks to develop techniques to modulate apoptotic activity in cancer cells by evaluating an electro-chemical approach to induce apoptosis.

Non-thermal atmospheric pressure dielectric barrier discharge plasma (Plasma) may provide a novel approach to induction of apoptosis. The purpose of this study was to evaluate the apoptotic effects of non-thermal plasma on human melanoma cells in vitro.

Methods: Melanoma cells were treated with increasing levels of non-thermal plasma in aluminum dishes by altering dose rate and evaluated by Trypan blue exclusion test, TUNEL analysis, Annexin-V/PI staining and Caspase-3 cleavage to determine viability and apoptotic activity at different time points from 3 hours to 72 hours post plasma treatment.

Figure 2. Schematic explaining the process of apoptosis in mammalian cells

Figure 1. Non-Thermal Plasma setup for treating melanoma cancer cells in-vitro.

 

 

Results: Trypan blue staining revealed that plasma treatment at low power for up to 5 seconds did not significantly increase the number of dead cells immediately following treatment (time zero); however, at higher doses, the percent dead cells increased linearly with dose of plasma. TUNEL analysis of cells treated for 15 seconds at high power demonstrated an increase in apoptosis at 24 and 48 hours post-treatment (p < 0.05). Annexin-V/PI staining revealed a significant increase in apoptosis in plasma-treated cells at 24, 48, and 72 hours post-treatment (p < 0.05).

 

Figure 6. Annexin staining confirmed no significant increase in apoptosis immediately after plasma treatment, but a significant increase in apoptosis in plasma-treated experimental cells at 24, 48, and 72 hours post-treatment (p < 0.001).

Figure 4. Untreated Melanoma Cancer Cells. All live cells are stained blue.

Figure 5. Data from triplicate samples (± S.D.) are expressed relative to total number of cells in untreated control. Total number of cells decreases 1 h, 3 h and 24 h after plasma treatment (p < 0.001). Also total number cells decreases linearly as the dose of plasma exposure increases from 5 J/cm2 to 30 J/cm

Figure 3. Melanoma cancer cells stained following TUNEL assay. All cells are stained blue and apoptotic cells are also stained green

 

 

 

 

 

 

Conclusions: Plasma treatment induces apoptosis in melanoma cells through a pathway that appears to be dependent on production of reactive oxygen species by plasma in fluid.  Since this plasma effect is non-thermal, this may be a selective way to treat cutaneous malignancies without initiating inflammatory responses. Non-thermal plasma may be a useful tool to induce directed cell death without inducing necrosis and inflammation.

Future Work: Future work will endeavour to better delineate the pathways that are triggered by sub-lethal Plasma exposure.  Evaluation of key signaling components of the apoptotic pathway will be performed.  The intrinsic and extrinsic pathway will be analyzed in order to determine how the non-thermal plasma induced effects lead to cell apoptosis.  Molecular arrays and real time PCR can identify target pathways and proteins respectively that are intimately involved in the cellular response to Plasma exposure.  A second avenue of research will focus on the actions of the various components of a Plasma discharge, isolating the Ultraviolet, the free radicals or the charged particles.

References

  1. Melanoma Death Rate Still Climbing

  2. Cancer Stat Fact Sheets

  3. Ries LAG, et al., eds. SEER Cancer Statistics Review, 1975–2000. Bethesda, MD: National Cancer Institute; 2003: Tables XVI-1-9.

  4. Parkin D, Bray F, Ferlay J, Pisani P (2005). "Global cancer statistics, 2002". CA Cancer J Clin 55 (2): 74–108.

  5. Lucas, R. Global Burden of Disease of Solar Ultraviolet Radiation, Environmental Burden of Disease Series, July 25, 2006; No. 13. News release, World Health Organization

  6.  Treatment of Skin Cancer.

  7. Fridman, G., Brooks, A. D., Balasubramanian, M., Fridman, A., Gutsol, A., Vasilets, V. N., Ayan, H.,  Friedman, G. 2006 Comparison of Direct and Indirect Effects of Non-Thermal Atmospheric Pressure Plasma on Bacteria, Plasma Process. Polym. 4: 370-375.

  8. Fridman, G., Peddinghaus, M., Ayan, H., Balasubramanian, M., Gutsol, A., Brooks, A. D., Fridman, A., Friedman, G. 2006. Blood Coagulation and Living Tissue Sterilization by Floating-Electrode Dielectric Barrier Discharge in Air, Plasma Chem. and Plasma Proc. 26(4): 425-442

  9. Fridman, G.,  A. Shereshevsky, M.M. Jost, A.D. Brooks, A. Fridman, A. Gutsol, V. Vasilets, G. Friedman. 2007. Floating Electrode Dielectric Barrier Discharge Plasma in Air Promoting Apoptotic Behavior in Melanoma Skin Cancer Cell Lines, Plasma Chemistry and Plasma Processing, 27(2): 163-176.

  10. Kieft, I. E., Darios, D., Roks, A. J. M. and Stoffels-Adamowicz, E., 2006. Reattachment and Apoptosis after Plasma-Needle Treatment of Cultured Cells, IEEE Tans. Plasma Sci. 34(4): 1331-1336

  11. Coulombe, S., Léveillé, V., Yonson, S. and Leask, R. L., 2006. Miniature atmospheric pressure glow discharge torch (APGD-t) for local biomedical applications. Pure and Applied Chemistry, 78(6): p. 1147-1156.

  12. Leveille, V. and S. Coulombe, 2005. Design and preliminary characterization of a miniature pulsed RF APGD torch with downstream injection of the source of reactive species. Plasma Sources Science and Technology, 14(3): p. 467(10).

  13. Coulombe, S. 2007. Live cell permeabilization using the APGD-t. In First International Conference on Plasma Medicine (ICPM-1). Corpus Christi, Texas.

  14. Kieft, I.E.   Darios, D.   Roks, A.J.M.   Stoffels, E., 2005. Plasma treatment of mammalian vascular cells: A quantitative description. Plasma Science, IEEE Transactions on, 33(2): 771-775.

  15. Kieft, I.E., M. Kurdi, and E. Stoffels, 2006. Reattachment and Apoptosis after Plasma-Needle Treatment of Cultured Cells. Plasma Science, IEEE Transactions on, 34(4):  1331-1336.

  16. Stoffels, E., 2006. Gas plasmas in biology and medicine. Journal of Physics D: Applied Physics, 39(16).

  17. Stoffels, E., Kieft, I. E., Sladek, R. E. J., Van den, L. J. M., 2006. Plasma needle for in vivo medical treatment: recent developments   and perspectives. Plasma Sources Science and Technology, 15(4):S169.

  18. G. Fridman, A. Shereshevsky, M. Peddinghaus, A. Gutsol, V. Vasilets, A. Brooks, M. Balasubramanian, G. Friedman, and A. Fridman, 2006. Bio-Medical Applications of Non-Thermal Atmospheric Pressure Plasma. In 37th AIAA Plasmadynamics and Lasers Conference. San Francisco, California.

  19. Shekhter, A.B., et al., 2005. Beneficial effect of gaseous nitric oxide on the healing of skin wounds. Nitric Oxide-Biology and Chemistry, 12(4): 210-219.

  20.  Gostev, V. and D. Dobrynin. 2006. Medical microplasmatron. In 3rd International Workshop on Microplasmas (IWM-2006). Greifswald, Germany.

  21. Kalghatgi, S. U., Fridman, G., Cooper, M., Nagaraj, G., Peddinghaus, G., Balasubramanian, M., Vasilets, V. N., Gutsol, A., Fridman, A., Friedman, G.  2007. Mechanism of Blood Coagulation by Non-Thermal Atmospheric Pressure Dielectric Barrier Discharge Plasma, IEEE Trans. Plasma Sci. 35(5): 1559-1566.

Relevant Publications, Talks and Posters

  1. Non-Thermal Plasma Promotes Apoptosis in Melanoma Cancer Cells, R. Sensenig, S. Kalghatgi, E. Podolsky, E. Cerchar,  G. Fridman, A. Shereshevsky, A. Fridman, G. Friedman, A. Brooks, J. Azizkhan-Clifford. (Manuscript to be submitted to the Journal of Experimental and Clinical Cancer Research).

  2. Mechanism of Induction of Apoptosis in Melanoma Cancer Cells by Non-Thermal Plasma, S. Kalghatgi, Ekaterina Cerchar, Gregory Fridman, Alexey Shereshevsky, Krishna Priya Arjunan, Rachel Sensenig, Erica Podolsky,  Behzad Torabi, Alexander Fridman, Ari Brooks,  Jane Azizkhan-Clifford, Gary Friedman, 36th IEEE International Conference on Plasma Science (ICOPS), May 29th - Jun 4th 2009, San Diego, California, USA.

  3. Induction of Apoptosis by Non-thermal Plasma Treatment of Melanoma Cancer Cells, S. Kalghatgi, Krishna Priya Arjunan, Rachel Sensenig, Ekaterina Cerchar, Erica Podolsky, Gregory Fridman, Behzad Torabi, Alexander Fridman, Ari Brooks,  Jane Azizkhan-Clifford, Gary Friedman, 2009 Drexel University Research Day, April 23rd 2009, Philadelphia, PA, USA

  4. Induction of Apoptosis by Non-thermal Plasma Treatment of Melanoma Cells, S. Kalghatgi, Krishna Priya Arjunan, Rachel Sensenig, Ekaterina Cerchar, Erica Podolsky, Gregory Fridman, Behzad Torabi, Alexander Fridman, Ari Brooks,  Jane Azizkhan-Clifford, Gary Friedman, 2nd International Conference on Plasma Medicine (ICPM-2), Mar 16th – Mar 20th 2009, San Antonio, Texas, USA

  5. Induction of apoptosis in Melanoma Cells by Non-Thermal Atmospheric Plasma Discharge, Rachel Sensenig, S. Kalghatgi,  Gregory Fridman, Alexey Shereshevsky, Ari Brooks, Victor Vasilets, Alexander Gutsol, Alexander Fridman, Gary Friedman, SSO's 61st Annual Cancer Symposium, March 13-16, 2008, Chicago. USA

  6. Induction of Apoptosis by Non-Thermal Plasma Treatment of Melanoma Cells, R Sensenig, S Kalghatgi, E Cerchar, B Torabi, E Podolsky, K Priya-Arjunan, G Fridman, A Fridman, G Friedman, A Brooks, J Clifford-Azizkhan, Drexel University College of Medicine Discovery Day, October 15th 2008, Philadelphia, PA.

  7. Non-Equilibrium Dielectric Barrier Discharge Plasma Promoting Apoptotic Behavior in Melanoma Skin Cancer Cells, G. Fridman, S. Kalghatgi, R. Sensenig, A. Shereshevsky, R. Stoddard, G. Friedman, A. Gutsol, V. Vasilets, A. Fridman, A.D. Brooks, NATO Advanced Study Institute (ASI), Plasma Assisted Decontamination of Biological and Chemical Agents, September 16-26, 2007, Çesme, Turkey

  8. Non-thermal atmospheric plasma induces apoptosis in melanoma cancer cells, A. Brooks, G. Fridman, R. Sensenig, A. Shereshevsky, L. Peddinghaus, M. Balasubramanian, M. Jost, D. Dobrynin, S. Kalghatgi, M. Cooper, A. Fridman, A. Gutsol, G. Friedman, First International Conference on Plasma Medicine (ICPM-1), October 15th - 18th, 2007, Corpus Christi, Texas. Presented by: Ari Brooks.

  9. Non-Equilibrium Dielectric Barrier Discharge Plasma Promoting Apoptotic Behavior in Melanoma Skin Cancer Cells, G. Fridman, S. Kalghatgi, A. Fridman, A. Gutsol, V. Vasilets, G. Friedman, R. Sensenig, A. Shereshevsky, M. Balasubramanian, M. Jost, A. Brooks, The 34th IEEE International Conference on Plasma Science, June 17-22, 2007, Albuquerque, New Mexico. Presented by: Gregory Fridman.

  10. Non-Equilibrium Dielectric Barrier Discharge Plasma Promoting Apoptotic Behavior in Melanoma Skin Cancer Cells, G. Fridman, R. Sensenig, S. Kalghatgi, A. Shereshevsky, M. Balasubramanian, A. Gutsol, V. Vasilets, A. Brooks, A. Fridman, G. Friedman, In Abstracts and full papers 18th International Symposium on Plasma Science, Aug 26th-Aug31st, Kyoto University, Kyoto, Japan : Kyoto University, 2007.

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