Finding a Way to Treat Cisplatin and Multiple Drug Resistant
Cancers
Shelly Jozwiak
The objective of this research was to determine if any of the new platinum and
palladium compounds synthesized by the Dr. Granger research team would kill cancer
cells that are resistant to cisplatin or multiple drugs in addition to killing cells that are not
resistant.
Background
Cancer remains an elusive disease for doctors in many cases and a scary diagnosis
for patients. One reason cancer is difficult to treat is that each type of cancer is different;
even the same type of cancer can vary from person to person in its cellular characteristics.
Two characteristics shared by all cancers are uncontrolled growth and invasiveness to
other parts of the body. Cancer is typically treated with surgery, radiation,
chemotherapy, immunotherapy, and/or gene therapy. While surgery and radiation are
ideally used for isolated tumors whose cells have not spread throughout the body, cancer
that has spread into other parts of the body is usually treated with one or more of the
other three options. One widely used chemotherapeutic agent is cisplatin, especially for
testicular, bladder, germ cell, head and neck, small cell lung cancer, and ovarian cancer.
Ovarian cancer is one of the four leading causes of death among women in the
United States. Over 80 percent of patients with ovarian cancer relapse after the first
treatment, and a majority of these develop resistance to the chemotherapy. Five year
survival rates for women diagnosed with this type of cancer for stages I, II, III, and IV are
74, 58, 30, and 19 percent, respectively. This would not be bad except for the fact that 74
percent of women with ovarian cancer are diagnosed in stages III and IV. The initial
response rates to chemotherapy (usually cisplatin-based) are high; cisplatin resistance is
the reason for the low survival rates. It is the limiting factor in chemotherapy treatment
of ovarian cancer. 50 percent of all ovarian cancers are intrinsically resistant to cisplatin
(having never been treated), so a woman diagnosed with ovarian cancer has a 50 percent
chance of even responding to the chemotherapy.
Resistance
Resistance occurs when, due to certain cell mechanisms, the cells are not affected
by a particular drug/substance. There are three different types of resistance: intrinsic,
acquired, and cross-resistance. Intrinsic resistance occurs when the cells are not affected
by the drug the very first time they come in contact with it. Acquired resistance occurs
after repeated treatments with a drug; a few resistant cells that are not killed by the first
few treatments grow and multiply until most of the cell population is now resistant.
Cross resistance occurs when cells that are resistant to one drug are also resistant to
another drug, usually by some similarity between the drugs.
How does a cell “resist” cisplatin? There are many ways: decreased intake,
increased efflux (output), enhanced DNA repair of damage caused by cisplatin, and
defective cell death pathways are some of the main mechanisms thought to account for
resistance. However, it is still unknown how much each of these reasons is responsible;
it does seem that there is usually a combination of two or more reasons.
Current attempts to circumvent cisplatin resistance have not resulted in large
increases in successful treatment. Treating the patients with compounds that reverse a
certain resistance mechanism increase response rates but only a small amount; this is
because resistance occurs by multiple mechanisms. The drugs used target only one
specific mechanism, such as a protein or enzyme that is found in levels too high or not
high enough. Combinations of drugs are also used that work more effectively together
than alone. However, the increases in response rates do not rise satisfactorily high.
Other drugs are constantly being tested, but none have been found yet that overcome all
of the current problems with cisplatin.
Goal and methods
The goal of this research was to determine if any new platinum and palladium
compounds were effective against cisplatin and multiple-drug resistant ovarian and
uterine cancer cells. The cells used include two cisplatin resistant ovarian carcinomas
(OVCAR-3 and SK-OV-3), two cisplatin resistant uterine sarcomas (MES-SA and its
multi-drug resistant derivative MES-SA/Dx5), an untreated ovarian carcinoma (MDAH
2774), a radiation treated uterine sarcoma (SK-UT-1), and a non-cancerous uterine line
(NUT).
The compounds used were those synthesized by Dr. R. Granger and his research
team of the Sweet Briar College chemistry department. These compounds were first
synthesized in 1996 by Dr. Granger and previously believed to be impossible to make.
Therefore, research involving these as possible anti-cancer agents has been very limited.
All of the compounds used in this particular study except for one were palladium
compounds.
Cells were grown in culture flasks in an incubator. When there was a sufficient
amount for an experiment, they were passed into well-plates, plastic plates with 96 small
wells. They were allowed to sit for a day, and the compounds were then added to the
wells for the cells to take up. Each well-plate had two controls, wells with untreated cells
and wells with cells treated with dimethyl sulfoxide (DMSO), which is used to dissolve
the compounds before adding them. After the addition of the compound, the cells were
allowed to sit for two days. The MTT assay was then used to assess cell survival rates.
Cell survival rates were measured on the basis of metabolic activity. A compound
called MTT was added at the end of the experiment. Cells that were alive would taker
the MTT up and metabolize it, and the dead cells would not. MTT in solution is yellow,
but it is a blue precipitate when metabolized by these human cells. Therefore, wells with
more living cells would have more blue precipitate than the wells with mostly dead cells.
MTT was added, and then the growth medium solution was removed four hours
later, leaving just the cells (adhered to the plastic) and metabolized MTT in the wells.
DMSO was put in the wells, which served to break open and kill the cells while
dissolving the MTT. The plates were then read in a microplate reader, which measured
the absorbance of each well at 540 nanometers. This was based on the use of absorbance
as a function of concentration—with higher metabolized MTT concentration, the
absorbance is higher. Therefore, a relative determination of the survival rates of the cells
(compared to the two controls) could be made.
Results and Discussion
After many weeks of testing (each experiment takes four days), results show that
almost all of these compounds kill the resistant as well as the non-resistant cell lines.
When compared with cisplatin, many of our compounds seem to be much more effective.
MDAH 2774, ovarian carcinoma
120
100
Pd(dione)Cl4
Pt(dione)Cl4
Pd(phen)Cl4
Pd(phen)2Cl2
Pd(dione)2Cl2
Pd(dione)(bipy)
Cisplatin
Pd(dione)(phen)
Pd(dppz)2
Pd(dione)Cl2`
Pd(dppz)(phen)
Percent survival
80
60
40
20
0
1
Compound
A comparison of the compounds used against MDAH 2774, an untreated ovarian carcinoma line. Cisplatin
was included in this study for comparison of effectiveness. Although MDAH 2774 was untreated, it turned
out to be intrinsically resistant to cisplatin.
Almost all of the compounds were more effective than cisplatin against all of the
cancerous cell lines tested. Several compounds consistently resulted in survival rates of
20 percent or below against all of the cell lines. Others were obviously more effective
than cisplatin as well, although not as effective against some cell lines as others.
The normal cell line was included in this study for added information about how
selective the compounds were against cancer cells vs. normal cells. The best outcome
would have been low survival rates against cancer cells and high survival rates against
normal cells. However, what we found most of the time was only a slight difference. A
few of the compounds, though, showed more selectivity than the others. Cisplatin did not
kill very many normal cells; it killed even less cancer cells, though. This proves, then,
that in many cases, more normal cells are killed than cancer cells during treatment.
There is more unknown about these specific compounds than there is known at
this particular point concerning their actions in cells. Exactly how the compounds
interact with the cells, in what way the cells are dying (accidental or programmed death),
why they work better than cisplatin, and if acquired resistance could be a problem is still
unknown. Further testing could shed some light on these questions.
Overall, the results show what was hoped for; these compounds show potential
for use as anti-cancer agents. However, this was only the first step. The next step
involves submitting our testing results and compound structural information to the
National Cancer Institute. They may then begin testing these compounds, first against a
panel of three cell lines and then against 60 cell lines; if these tests are promising, clinical
trials will then begin.