Naranamangalam R Jagannathan1, Khushbu Agarwal1, Uma Sharma1, Sandeep Mathur2, Vurthaluru Seenu3, and Rajinder Parshad3
1Department of NMR and MRI Facility, All India Institute of Medical Sciences, New Delhi, India, 2Department of Pathology, All India Institute of Medical Sciences, New Delhi, India, 3Department of Surgical Disciplines, All India Institute of Medical Sciences, New Delhi, India
Synopsis
We
evaluated the changes in tCho levels
and β-catenin expression (membrane and cytoplasm) after III neoadjuvant
chemotherapy in breast cancer patients. Significant reduction in β-catenin expression
(membranous and cytoplasmic) was observed after therapy. Post-therapy, tCho
reduced significantly in tumors with Grades 1 and 2 membranous
β-catenin expression and also in tumors with
IRS 0 and Grade 1 cytoplasmic β-catenin. Prior to therapy, tCho was positively
associated with cytoplasmic β-catenin while negatively with membranous protein.
However post-therapy tCho was negatively associated with both cytoplasmic and
membranous β-catenin. This signifies antiproliferative and apoptosis induction
effects of chemotherapy drugs on breast cancer patients.Purpose
To determine
the effect of chemotherapy on the expression of membranous and cytoplasmic β-catenin
and tCho concentration in breast cancer patients
Methodology
The
present study was undertaken to explore the changes associated with neoadjuvant
chemotherapy (NACT) treatment on levels of tCho and expression of β-catenin (membrane
and cytoplasm) in breast cancer patients. Twenty
patients with locally advanced breast cancer (LABC) were recruited for in vivo
1H
magnetic resonance spectroscopy (MRS) at 1.5 T (Avanto, Siemens). Table 1
presents the clinical details of the patients. tCho concentration was
calculated using the integral values
1 of the acquired MR spectrum
from each patient both prior to and after III NACT. Tissues (biopsy before NACT
and surgical specimen after NACT) obtained from the same set of patients were
immediately fixed in formalin and paraffin embedded blocks were prepared. Immunohistochemistry
(IHC) was carried out using anti-β-catenin antibodies. The intensity was scored
as follows: 0 = none, 1+ = weak, 2+ = moderate, and 3+ = strong/intense. The percentage
of β-catenin positive cells was scored from 1 to 7 (1 = 0%; 2 = 1–4%; 3 =
5–19%; 4 = 20–39%; 5 = 40–59%; 6 = 60–79%; 7 = 80–100%). Immunoreactive scores
(IRS) were calculated by multiplying the scores for percentage and intensity
2.
Patients with IRS of 3-8 were categorized as Grade 1 and IRS of 9-21 as Grade
2. Wilcoxon matched pairs two tailed test and Spearman’s rank analysis were used
to calculate the significance and correlations. A p-value of ˂0.05 was
considered significant. Institutional ethical committee approved the study and
written informed consent were obtained from all patients.
Results and discussion
In
the present study, the effect of chemotherapy on the expression of β-catenin in
both membrane and cytoplasm as well as on tCho concentration was investigated
in breast cancer patients. Table 2 presents the grades based on IRS for expression
of β-catenin (membrane and cytoplasm) and the tCho concentration both prior to
and after III NACT. Figure 1a shows the positive control (Split end, SPEN) and Figs.
1b-e are the representative slides showing β-catenin expression in cytoplasm
and membrane both pre- and post-therapy. Prior to therapy, membranous
immunoreactivity to β-catenin was seen in 18/20 (90%) patients; of these 15%
showed Grade 1 while 75% patients had Grade 2 staining. Cytoplasmic
immunoreactivity to β-catenin was seen in 10/20 (50%) patients prior to
therapy; of these, 15% showed Grade 1 while 35% patients showed Grade 2
staining. After III NACT, both membranous and cytoplasmic expression of
β-catenin reduced significantly (Figure 2; Table 2). Further, our data showed
that the concentration of tCho significantly reduced following chemotherapy
compared to its pre-therapy value in tumors with membranous expression of
β-catenin of Grade 1 and Grade 2 (Table 2). The tumors with cytoplasmic
β-catenin expression in IRS 0 and Grade 1 also showed a significant reduction
in tCho concentration after therapy (Table 2). The decrease in tCho
concentration observed may be due to the cytotoxic activity of chemotherapy
drugs. This may be related to decrease in the activity of major enzymes
involved in choline synthesis pathway like choline kinase, phospholipase D
(PLD) and phospholipase A2
3. Prior to therapy, a positive
association between tCho concentration and cytoplasmic β-catenin while a
negative correlation with membranous β-catenin was observed which may be attributed
to the translocation of β-catenin from membrane to cytoplasm. The increased
levels of β-catenin in cytoplasm increases the activity of PLD, which in turn
enhances the tCho concentration as observed prior to therapy. However,
post-therapy a negative association of tCho with both cytoplasmic and
membranous β-catenin was observed. The antiproliferative and apoptosis
induction effects of chemotherapy drugs mediate the suppression of Wnt/β-catenin
signaling, and these actions might contribute to a decrease in β-catenin
expression in membrane and cytoplasm of the cells. This leads to decreased activity
of PLD thereby decreasing the tCho concentration after NACT in breast cancer
patients.
Conclusion
In
the present study the association between tCho concentration and the
cytoplasmic and membranous β-catenin emphasizes the possibility that both tCho
and β-catenin influence breast cancer progression. While their decrease
followed by NACT treatment reflects the cytotoxic effect of chemotherapy drugs
on these molecular pathways in these LABC patients. The results of our present
study call for additional studies to determine the mechanism of NACT effect on
different molecular pathways involved in breast cancer.
Acknowledgements
The authors thank the Department of Science and Technology, Government of India for the financial assistance.References
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