Abstract
Background/Aim: Platinum-based chemotherapies are a component of standard-of-care regimens for urothelial carcinoma (UC). These nephrotoxic drugs are often dose-limiting, with cisplatin and carboplatin being the most commonly used. Dicycloplatin (DCP) has better solubility and stability, with comparable efficacy and better tolerability. Some suggest the use of DCP as primary treatment for non-muscle-invasive bladder cancer. We exposed UC cell lines to DCP in vitro to assess its efficacy. Materials and Methods: A high grade (IV) in vitro UC cell line (TCCSUP) was exposed to varying concentrations of cisplatin (0-600 μg/ml), carboplatin (0-600 μg/ml), oxaliplatin (0-4.0 μg/ml), and DCP (0-350 μg/ml). Grade II-IV cells were exposed to varying concentrations of DCP (0-350 μg/ml) to assess time- and concentration-dependent growth inhibition, and simulate intravesical treatment. Growth inhibition was determined following 24, 48, and 72 h of exposure, using a tetrazolium dye to assess mitochondrial dehydrogenase activity. Results: DCP, cisplatin, and carboplatin effectively achieved >90% cell kill at 72 h. Concentrations of 325 μg/ml DCP, 50 μg/ml cisplatin, and 600 μg/ml carboplatin were sufficient for >90% cell-kill, with cisplatin demonstrating the highest efficacy at the lowest concentration/time intervals. Dose- and time-dependent cell kill were demonstrated at varying concentrations of DCP in grade II-IV cell lines, including cells exposed intravesically. Conclusion: In vitro, DCP demonstrates cell-killing efficacy in a time- and concentration-dependent manner in grade II-IV UC cell lines, showing promise for its intravenous, oral, and intravesical use for bladder UC in both primary and adjuvant/neoadjuvant settings.
Urothelial carcinoma (UC) is extremely prevalent, with an estimated 84,825 new cases in the United States in 2022, 65,181 of which were men, and 19,223 deaths (1). UC consistently ranks in the top ten most commonly diagnosed cancers both worldwide and in the United States, but UC prevalence is about threefold higher in Europe and North America (2). In the United States, lifetime risk of UC is 3.9% in men and 1.2% in women, respectively (2). Five-year survival in Europe and the United States approaches 70%, with notable divergence based on the involvement of the detrusor muscle (2). The leading cause of bladder cancer is carcinogen exposure, with cigarette smoking accounting for approximately 66% of male cancers and 30% of female cancers. Smokers boast a two- to four-fold increased risk when compared to non-smokers (3, 4). Presenting symptoms often include hematuria (up to 85%) and lower urinary tract symptoms, such as dysuria and urinary frequency (5). Approximately 10% of patients with gross hematuria and 2-5% of patients with microscopic hematuria will be diagnosed with bladder cancer (6). Approximately 60-80% of newly diagnosed bladder cancers are superficial, either Ta, T1, or carcinoma in situ (Cis) (7). With local resection and no adjuvant intravesical therapy, progression rates for tumor grades I, II, and III and stage Ta/T1 to muscle-invasive cancer are 2, 11, and 45%, respectively (8).
Treatment of UC diverges based on involvement of the detrusor muscle in pathologic specimens. Non-muscle-invasive disease (NMIBC) treatment involves periodic surveillance, but if it is intermediate-to-high-risk, can include intravesical chemotherapy and immunotherapy with gemcitabine, doxorubicin, mitomycin-c (MMC), or Bacillus Calmette Guerin (BCG).
BCG is the current gold standard, with historic data showing decreased recurrence in superficial bladder cancer when compared to transurethral resection alone (9). Costs associated with bladder cancer are substantial, surpassing $6 billion in 2020 (10). The cost of BCG and its intravesical administration and follow up, for one year of treatment (induction and maintenance) per patient, has been estimated to be about $29,459 per patient in the US, or about $373 million for all treated US patients annually (10). After 2019, there have been recurring shortages and supply issues with obtaining BCG for UC patients, and subsequent intravesical chemotherapies are attempting to replace BCG.
Gemcitabine, doxorubicin, epirubicin, and MMC have all shown benefit in reducing UC recurrence when administered intravesically in the immediate post-operative setting following transurethral resection of bladder tumor (TURBT) (11). However, BCG is the only known agent to decrease risk of progression from NMIBC to MIBC (12).
In the modern era, adjuvant therapy options diverge based on risk stratification per the American Urological Association (AUA) (12). NMIBC patients with low-to-intermediate risk disease often receive one of the above regimens in the immediate post-operative setting without the addition of induction therapy. However, NMIBC intermediate-risk patients can consider, and NMIBC high-risk patients should utilize, induction chemotherapy or immunotherapy.
Systemic therapy and radical surgery are reserved for NMIBC patients resistant to endoscopic resection and intravesical therapy. However, the gold-standard for treatment of muscle-invasive disease is radical cystectomy, often preceded by neoadjuvant platinum-based chemotherapy (NAC). Systemic NAC, particularly platinum-based, can be harsh, nephrotoxic, and often treatment-limiting, with renal dysfunction and ototoxicity cited as common factors for patients not receiving NAC (13). However, the benefits of treating micro-metastatic disease are continually demonstrated by increased overall survival in patients receiving NAC when compared to radical cystectomy alone, with approximately 5% survival benefit at five years (14, 15). Intravenous administration is required with currently-approved platinum regimens, leading to increased treatment complexity and burden.
Dicycloplatin (DCP), a new platinum analog, can be administered both intravesically and systemically, with oral bioavailability. DCP may change the treatment paradigm of UC in both the NMIBC and MIBC UC setting. DCP appears less nephrotoxic than other platinum drugs, as evidenced anecdotally in a patient who self-treated off-label at our center, and supported by preliminary data form China (16, 17). Although DCP has not been studied extensively in UC, it has already shown promise in other platinum-sensitive malignancies, including non-small cell lung cancer (NSCLC). In Chinese trials in NSCLC patients comparing carboplatin to DCP, both in combination with paclitaxel, median progression-free survival (4.1 vs. 4.9 months) and overall three year survival (11.1% vs. 22.2%) were prolonged in the DCP group with no difference in adverse reactions between groups (18).
The above data have led us to hypothesize that DCP will inhibit the growth of tumorigenic bladder cells in vitro, prove effective against transplantable bladder cancer cell lines in immune-deficient mice, and ultimately be a potentially effective form of intravesical and systemic therapy for human bladder cancer.
Materials and Methods
Tissue culture media. RPMI 1640 tissue culture media were supplemented with glucose, L-Glutamine, HEPES, 10% Fetal Bovine Serum (ATCC, Manassas, VA, USA) and 1% penicillin streptomycin. This medium was used for all cell passage and experimental procedures.
Platinum drugs. Dicycloplatin (DCP) was manufactured in Beijing, China. DCP was dissolved in sterile water to 0.5 mg/ml. Subsequent dilutions to 0-100 μg/ml were made in tissue culture media. Cisplatin (Fisher Scientific, Pittsburgh, PA, USA) was reconstituted in 154 mM NaCl and diluted to 0-600 μg/ml. Carboplatin (Sigma-Aldrich, St. Louis, MO, USA) was solubilized in sterile water to concentrations of 0-600 μg/ml. Oxaliplatin (donated by Mary Babb Randolph Cancer Center) was diluted in sterile water to concentrations of 0-4.0 μg/ml. Human bladder cancer cell lines were exposed to varying concentrations of platinum drugs as listed above to assess the time and concentration-dependence of growth inhibition. Tissue culture media alone served as the control for all experimental procedures.
Cell culture and reagents. Three human bladder cancer cell lines, TCCSUP (Grade IV), T24 (Grade III) and HTB9 (Grade II) (ATCC) were maintained as monolayers in their preferred media (as described above) at 37°C in 5% CO2. Cells were trypsinized and then plated in sterile 96-well plates at 1×105 cells/ml. Cells were then returned to the incubator for 24 h to allow adherence prior to exposure to platinum drugs. Cells were treated with platinum drugs at different concentrations and returned to the incubator for an additional 24, 48, and 72 h. In the intravesical simulation assay, cells were treated as above for two hours. Subsequently, they were washed and exposed to their preferred medium for 24, 48, and 72 h.
MTT assay. The MTT colorimetric assay was performed to detect tumor cell viability after 24, 48 and 72 h of incubation. MTT, a tetrazolium dye (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; thiazolyl blue (Sigma-Aldrich) was added to each well as described previously (19). Plates were incubated in the presence of MTT dye for 4 h. Mitochondrial dehydrogenase activity reduced the yellow MTT dye to a purple formazan, which was then solubilized with acidified isopropanol and absorbance was read at 570 nm.
Statistical analysis. Determination of statistical significance was performed by analysis of variance (ANOVA) (20). Post hoc comparison of individual concentration means with the control was completed using the Tukey-Kramer Multiple Comparison test (21). All data are reported as means±SD.
Results
Increasing concentration of DCP. HTB9 (grade II), T24 (grade III), and TCCSUP (grade IV) cell lines demonstrated concentration- and time-dependent growth inhibition when exposed to DCP in vivo. At maximal concentration (350 μg/ml) and 72 h exposure, near 100% cell kill was demonstrated in all cell lines. Specifically, HTB9, T24, and TCCSUP exhibited 96.15, 94.24, and 90.17% growth inhibition, respectively, with 0% growth inhibition observed in the control. When analyzed for concentration dependence, all groups reached significance (p<0.0001) and demonstrated the expected increase in growth inhibition with increasing concentrations of DCP, which ranged incrementally from 20 to 350 μg/ml. When considered individually, HTB9 cells showed a significant difference in growth inhibition from the control at all concentrations above 125 μg/ml (aside from 200 μg/ml), 25 μg/ml (aside from 125 and 150 μg/ml), and 20 μg/ml at 24, 48, and 72 h, respectively. T24 cells reached a significant difference in growth inhibition from the control at all concentrations above 225 μg/ml, 20 μg/ml, and 20 μg/ml at 24, 48, and 72 h, respectively. TCCSUP cells reached a significant difference in growth inhibition from the control at 200 μg/ml, all concentrations above 20 μg/ml, and 20 μg/ml at 24, 48, and 72 h, respectively (Table I).
HTB9 (grade II), T24 (grade III), and TCCSUP (grade IV) urothelial carcinoma growth inhibition at varying concentrations of dicycloplatin (DCP) and at different time points.
Intravesical simulation. Similarly, HTB9 (grade II), T24 (grade III), and TCCSUP (grade IV) cell lines demonstrated concentration- and time-dependent growth inhibition when exposed to DCP in vitro for two hours, followed by washing and growth in their preferred medium (Table II). The maximum mean growth inhibition demonstrated occurred in the TCCSUP cell line at 72 h (39.26%). When analyzed for concentration dependence, all groups reached significance (p<0.0001) and demonstrated the expected increasing growth inhibition at increasing concentrations of DCP, incrementally increased from 100 to 350 μg/ml. When considered individually, HTB9 cells showed a significant difference in growth inhibition from the control at 350 μg/ml, all concentrations above 100 μg/ml, and 100 μg/ml at 24, 48, and 72 h, respectively. T24 cells failed to show a difference in growth inhibition from the control at 24 h, but reached a significant difference in growth inhibition from the control at all concentrations above 225 μg/ml and 225 μg/ml at 48 and 72 h, respectively. Similarly, TCCSUP cells failed to show a difference in growth inhibition from the control at 24 h, but reached a significant difference in growth inhibition from the control at all concentrations above 100 μg/ml and 100 μg/ml at 48 and 72 h, respectively (Table II).
HTB9 (grade II), T24 (grade III), and TCCSUP (grade IV) urothelial carcinoma growth inhibition at varying concentration of dicycloplatin (DCP) and at different time points after two hours exposure.
Other platinum analogs. Concentrations utilized for other platinum analogs varied based on known cell growth inhibition. In this assay, TCCSUP (grade IV) cell lines demonstrated concentration- and time-dependent growth inhibition when exposed to increasing concentrations of the respective platinum analogs in vitro. When analyzed for concentration dependence, all groups reached significance (p<0.0001) and demonstrated the expected increasing growth inhibition at increasing concentration when exposed to DCP (100-350 μg/ml), cisplatin (50-600 μg/ml), carboplatin (50-600 μg/ml), and oxaliplatin (0.5-4 μg/ml). The maximal mean growth inhibition occurred in the group treated with highest concentration of cisplatin (600 μg/ml; 72 h (95.44%). When considered individually, TCCSUP cells exposed to DCP reached a significant difference in growth inhibition from the control at 300 μg/ml, all concentrations above 100 μg/ml, and 100 μg/ml at 24, 48, and 72 h, respectively. TCCSUP cells exposed to cisplatin reached a significant difference in growth inhibition from the control at all concentrations above 50 μg/ml at 24, 48, and 72 h, respectively. TCCSUP cells exposed to carboplatin reached a significant difference in growth inhibition from the control at all concentrations above 400 μg/ml, 50 μg/ml and 50 μg/ml at 24, 48, and 72 h, respectively. TCCSUP cells exposed to oxaliplatin showed a significant difference in growth inhibition from the control at all concentrations above 2 μg/ml, 0.6 μg/ml, and 0.5 μg/ml at 24, 48, and 72 h, respectively (Table III).
TCCSUP (grade IV) urothelial carcinoma growth inhibition at varying concentrations of dicycloplatin (DCP), cisplatin, carboplatin, and oxaliplatin and time.
Discussion
The present BCG shortage has led to a race to find alternative therapies for use in intermediate- and high-risk NMIBC. Combination therapy with intravesical gemcitabine and docetaxel provides promise, with short-term studies indicating equivalent or superior survival compared to BCG (22). Herein, we present the first cell-line data for the platinum analog DCP, which may have a role in both NMIBC and muscle-invasive disease. It is the only platinum analog with both oral and intravenous bioavailability and is effective in vitro when simulated intravesically. The advantages of therapy with DCP may be multiple: 1) provision of an alternative intravesical therapy, 2) serving as an oral platinum analog for both the NMIBC and neoadjuvant settings, and 3) providing another intravenous platinum-based chemotherapy option, which is less nephrotoxic than cisplatin or carboplatin.
Our interest in DCP for UC was initially peaked by one of our own patients who received therapy in China. A 65-year-old man was initially diagnosed with intermediate-risk NMIBC in 2016. He underwent TURBT and was offered induction BCG therapy. He declined BCG and travelled to Beijing, China, where he received 300 mg IV DCP weekly for eight weeks. He also used oral DCP 12 mg DCP three times daily for six weeks. He also received yearly “booster” doses of DCP, which consisted of 300 mg IV weekly for two weeks. He underwent periodic surveillance cystoscopy at our institution, with no evidence of tumor recurrence (12). After five years, he was unable to travel to China due to the COVID-19 pandemic, and a small tumor recurrence at the prior resection site was noted. The patient received 27 mg orally daily for 10 days, and then 36 mg orally daily for five weeks. On cystoscopy at our hospital, the papillary tumor at the prior resection site was no longer present (16).
Although anecdotal, the above case demonstrates the clinical efficacy and safety of DCP. DCP has received blanket approval for “solid tumors” in China, with supporting evidence published in the Chinese literature. It has demonstrated comparable efficacy to lobaplatin and heptaplatin in cell-line studies performed similarly to the above in resistant colon cancer (23). In a phase I clinical trial in patients with various solid tumors who had failed established therapy, dose-escalation from 50 mg/m2 to 550 mg/m2 was performed. Of the 20 patients with measurable disease, two had confirmed partial response, 13 remained stable, and five showed disease progression (24). However, given the phase I nature of the trial, clinical safety was the preliminary endpoint. Grade 3-4 treatment-related adverse events were rare, with two patients (6.8%) experiencing dose-dependent, reversible thrombocytopenia, two patients (6.8%) experiencing dose-dependent, reversible anemia, and six patients (20.7%) experiencing nausea and vomiting resistant to anti-emetic therapy. The recommended starting dose for phase II clinical trials was 450 mg/m2, which was utilized in the aforementioned NSCLC trial in combination with paclitaxel (18, 24).
Tolerability of DCP has been demonstrated in vivo as well. Yu et al. quantified the adverse effects of DCP, cisplatin, and carboplatin in mice injected with ½ LD50 intraperitoneally daily for seven days. Those treated with DCP showed less bone marrow apoptosis, fewer arrested splenocytes, and less suppression of CD8 T cells and CD8 memory T cells (25). In human hepatoma and lung cancer cells lines, DCP inhibited proliferation, induced apoptosis (via both the death receptor pathway and mitochondrial pathway), up-regulated p53, and changed the mitochondrial membrane potential in a concentration-dependent manner (26).
Our in vitro data, reported here, confirm the previously mentioned reports regarding the antineoplastic potential of DCP and appear to be the first reported in UC. However, many DCP study limitations remain to be addressed. Although DCP has been studied in humans in China, the optimal dosing and route of administration for US patients have yet to be established. DCP’s safety has been demonstrated in solid organ tumors in Chinese studies. However, DCP oral pharmacokinetics are largely uncharacterized. Thus, studying DCP in both oral and intravenous formulations in an animal model specific to UC will be helpful prior to moving forward with human trials.
Conclusion
The orally bioavailable platinum analog DCP provides reliable time- and concentration-dependent cell kill/growth inhibition against UC cell lines in vitro. An oral platinum analog like DCP provides promise in both the neoadjuvant and adjuvant setting for the treatment of UC, particularly when comparable to the parenterally-administered analogs cisplatin, carboplatin, and oxaliplatin. Similarly, we demonstrated that exposure to the antineoplastic agent for shorter periods, such as during intravesical instillation, also effectively inhibits growth in UC cell lines.
Our in vitro data, reported herein, confirm the antineoplastic potential of DCP as reported in Chinese studies and represent the first report focused on DCP efficacy against UC in vitro. We now plan to study DCP in an animal model of transplantable UC, before proceeding to phase I clinical trials.
Acknowledgements
No outside help was obtained from individuals aside from the listed Authors.
Footnotes
Authors’ Contributions
David Zekan: Participated in research design, data analysis, and writing of paper. Thomas Hogan, Stanley Kandzari: Participated in research design and writing of paper. Barbara Jackson, Garrett Jackson: Participated in research design and performance of research.
Conflicts of Interest
All Authors declare that they have no competing interests, financial or non-financial, to disclose.
Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
- Received May 21, 2024.
- Revision received June 8, 2024.
- Accepted June 10, 2024.
- Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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